WO2011133658A1

WO2011133658A1 - Compositions and methods for targeting and delivering therapeutics into cells

Info

Publication number: WO2011133658A1
Application number: PCT/US2011/033229
Authority: WO
Inventors: John R. Murphy
Original assignee: Boston Medical Center Corporation
Priority date: 2010-04-22
Filing date: 2011-04-20
Publication date: 2011-10-27

Abstract

The invention features compositions for deliverying cytotoxic, therapeutic, diagnostic agents to a targeted cell and methods of using these compositions for treating disease.

Description

COMPOSITIONS AND METHODS FOR TARGETING AND

DELIVERING THERAPEUTICS INTO CELLS

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

This invention was made with Government Support under Contract Nos.

AI021628 and A057159 awarded by the National Institutes of Health. The

Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions that target and deliver therapeutics through the cell membrane for the treatment of disease or disorders and methods of use thereof.

BACKGROUND OF THE INVENTION

Diphtheria toxin (DT) (58 kDa) is a typical single chain AB toxin composed of three functional domains: the amino terminal catalytic (C) domain ("C-domain") corresponds to fragment A (21 kDa), and the transmembrane (T) and carboxy terminal receptor binding (R) domains comprise fragment B (37 kDa) of the toxin (Choe et al, Nature 357: 216-22, 1992). A disulfide bond between Cysl 86 and Cys201 subtends a protease sensitive loop and connects fragment A with fragment B. Furin mediated cleavage within this loop and retention of the disulfide bond have been shown to be prerequisites for intoxication of eukaryotic cells (Tsuneoka et al, J Biol. Chem. 268:26461- 5, 1993; Ariansen et al, Biochem. 32:83-90, 1993). Substitution of the native R domain with human interleukin-2 (IL-2) and other receptor binding domains has resulted in the formation of a fusion protein toxin whose cytotoxic action is specifically targeted only to cells expressing receptors capable of binding the receptor binding domain (see, e.g., Bacha et al, J. Exp. Med. 167:612-622, 1988; Waters et al, Eur. J. Immunol. 20:785-91, 1990; Ratts and vanderSpek, Diphtheria Toxin: Structure Function and its Clinical Applications. In Chimeric Toxins, H. Lorberboum-Galski, P. Lazarovici, eds., Taylor and Francis, London, New York. p. 14-36, 2002).

The intoxication of eukaryotic cells by DT follows an ordered series of interactions between the toxin and the cell which leads to inhibition of protein synthesis and cell death (Love and Murphy, Gram-Positive Pathogens, American Society for Microbiology, Washington, D.C., V.A. Fischetti, J. Rood Ed. pp. 573-582, 2000). The intoxication process is initiated by the binding of the toxin to its cell surface receptor, a heparin binding epidermal growth factor-like precursor and CD9. Once bound to its receptor, the toxin is internalized by receptor-mediated endocytosis into an early endsosomal compartment (Moya et al., J. Cell. Biol, 101 :548, 1985). Upon acidification of the endosomal lumen by vesicular (v)-ATPase, the T-domain undergoes a confonnational change and spontaneously inserts into the vesicle membrane forming an 18 - 22 A pore or channel (Kagan et al., Proc. Natl. Acad. Set, USA, 78:4950, 1981; Donovan et al., Proc. Natl. Acad. Set, USA, 78:172, 1981). The C-domain, in a fully denatured form, is then specifically thread through this channel and released into the cytosol. Once the C-domain is refolded into an active conformation it catalyzes the NAD⁺-dependent ADP-ribosylation of elongation factor 2 (EF-2), causing irreversible inhibition of protein synthesis and death of the cell by apoptosis (Pappenheimer, Annu. Rev. Biochem. , 46:69, 1977; Kochi and Collier, Exp. Cell. Res. , 208:296, 1993).

In recent years the study of the molecular mechanism by which the DT C- domain is translocated across the endosomal membrane and released into the eukaryotic cell cytosol has been the focus of much attention (Lemichez et al., 1997; Oh et al, 1999; Ren et al, 1999; Ratts et al, 2003; Ratts et al, 2005). In 2005, Ratts et al. conducted an in silico analysis of DT which described the Tl motif in helix 1 of the transmembrane domain. This motif was found in anthrax Lethal Factor (LF) and Edema Factor (EF), and botulinum neurotoxins serotypes A, C, and D; wherein its location is conserved in regions of these toxins that are believed to first emerge through a trans-endosomal membrane pore on the cytosolic side of the vesicle during the catalytic domain entry process.

COPI is a heptameric complex ( , β, β', γ, ε, ζ and δ subunits) which

facilitates vesicular retrograde transport between Golgi compartments, the Golgi apparatus and the ER, and in endosomal vesicle trafficking (Waters et al., 1991 ;

Serafini, et al., 1991; Whitney, et al, 1995). COPI complexes are recruited to the membrane surface en bloc by Arf-GTP (Donaldson et al, 1992; Palmer et al, 1993). Following the initial binding of COPI to the membrane, additional interactions through secondary binding of the complex to dibasic signatures (KKXX, KXKXX) and/or aromatic amino acid sequences (i.e. FFXXBB(X)„,) that are present in the cytoplasmic tails of cargo and p23/24 adaptor proteins further stabilize binding to the membrane surface (Cosson & Letourneur, 1994; Harter & Wieland, 1998; Eugster et ah, 2004). The requirements for C-domain translocation of DT and related toxins across endosomal membranes have been partially defined in PCT patent application publication number WO2005014798 and in U.S. Patent Application Publication No. US 2008-0306003 Al, both of which are incorporated herein by reference in their entirety.

RNA interference (RNAi) is the use of short dsRNA molecules whose sequence matches that of a gene of interest to selectively silence the expression of a gene in the genome. Once in a cell, a dsRNA molecule is cleaved into segments approximately 22 nucleotides long, called short interfering RNAs (siRNAs). siRNAs become bound to the RNA-induced silencing complex (RISC), which then also binds any matching mRNA sequence present in the cytosol. Once this occurs, the mRNA is degraded, effectively silencing the gene from which the mRNA was transcribed.

RNAi compositions hold promise as powerful therapeutics owing to the exquisite selectivity of RNAi: an RNAi sequence seeks out and silences its target without affecting the expression of other genes. RNAi can be applied to treat diseases and disorders caused by the expression of a diverse set of genes, e.g., viral genes, oncogenes, and genes responsible for heart disease, Alzheimer's disease, diabetes, and others.

To be a successful drug, an RNAi molecule must be stable enough to reach its target cells before it is degraded or excreted; it must get into those cells and hybridize with its intracellular target; and it must exert enough of an effect to improve the health of the patient to which the RNAi molecule is administered without causing significant toxic effects in either target or nontarget tissues. At present, delivery, stability, and potency have been the most significant obstacles. Thus, there exists a need for therapeutics capable of delivering nucleic acid (e.g., RNAi molecules) and amino acid therapeutics that are effectively targeted and delivered to specific cells and able to treat diseases or disorders caused by those cells.

SUMMARY OF THE INVENTION

In a first aspect, the invention features a conjugate compound having the general formula:

X-Y-Z (Formula I), wherein: X is selected from a cytotoxic agent, a therapeutic agent, and a diagnostic agent and comprises at least one thiol-containing group capable of forming a disulfide bond with Y;

Y is a polypeptide comprising one or more lysine-rich domains that are capable of interacting with cellular COPI complex proteins and that includes at least cysteine residue capable of forming a disulfide bond with the thiol-containing group of X; and

Z is a polypeptide targeting moiety that is bound to Y at its carboxy-terminal end. In an embodiment, X is selected from a siRNA, dsRNA, an RNAi molecule, a protein nucleic acid (PNA) molecule, and a polypeptide (e.g., a transcription factor or growth factor). In another embodiment, the lysine-rich domain includes one or more lysine rich motifs having a dibasic signature selected from KKXX and KXKXX, or aromatic amino acid sequences selected from FFXXBB(X)„,. In yet another embodiment, Y includes an amino acid sequence having at least 80% (e.g., 85%, 90%, 95%, 99%, or 100%») sequence identity to a contiguous amino acid sequence corresponding to at least amino acids 201 to 235 (e.g., amino acids 201 to 389 or amino acids 195 to 389) of diphtheria toxin. In yet another embodiment, the thiol-containing group of X is a cysteine residue that forms a disulfide bond with a cysteine residue of Y. In other embodiments, X is a nucleic acid molecule and the thiol-containing group is located at the 3' or 5' end of X or X is a polypeptide and the thiol-containing group is located at the amino- or carboxy-terminal end of X. In still other embodiments, Z is an antibody or is selected the group consisting of insulin, insulin-like growth factor receptor 1 (IGF1R), IGF2R, insulin- like growth factor (IGF; e.g., IGF 1 or 2), mesenchymal epithelial transition factor receptor (c-met; also known as hepatocyte growth factor receptor (HGFR)), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), heregulin, fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), platelet-derived growth factor (PDGF), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor (VEGF), tumor necrosis factor receptor (TNFR), tumor necrosis factor alpha (TNF-ct), TNF-β, folate receptor (FOLR), folate, transferring, transferrin receptor (TfR), mesothelin, Fc receptor, c-kit receptor, c-kit, an integrin (e.g., an a4 integrin or a β-l integrin), P-selectin, sphingosine- 1 -phosphate receptor- 1 (SI PR), hyaluiOnate receptor, leukocyte function antigen- 1 (LFA- 1 ), CD4, CD 1 1 , CD18, CD20, CD25, CD27, CD52, CD70, CD80, CD85, CD95 (Fas receptor), CD 106 (vascular cell adhesion molecule 1 (VCAM1), CD 166 (activated leukocyte cell adhesion molecule (ALCAM)), CD178 (Fas ligand), CD253 (TNF-related apoptosis-inducing ligand (TRAIL)), ICOS ligand, CCR2, CXCR3, CCR5, CXCL12 (stromal cell-derived factor 1 (SDF-1)), interleukin 1 (IL-1), IL-lra, IL-2, IL-3, IL-4, IL- 6, IL-7, IL-8, CTLA-4, MART-1, gplOO, MAGE-1, ephrin (Eph) receptor, mucosal addressin cell adhesion molecule 1 (MAdCAM-1), carcinoembryonic antigen (CEA), Lewis^Y, MUC-1, epithelial cell adhesion molecule (EpCAM), cancer antigen 125 (CA125), prostate specific membrane antigen (PSMA), TAG- 72 antigen, erythroblastic leukemia viral oncogene homolog (ErbB) receptor, and fragments thereof. In yet another embodiment, the invention features an interference-nucleotide as the "cargo" portion of the conjugate compound, such as, for example an iRNA or siRNA, which is adapted to inhibit or decrease transcription or translation of a factor(s) that is part of the CTF complex (e.g., β-COP, γ-COP, Hsp-90, and TrR-1).

A second aspect of the invention features a method of treating disease in a mammal in need thereof by administering the conjugate compound of the first aspect of the invention to the mammal (e.g., a human). In an embodiment, the conjugate compound includes a cytotoxic or therapeutic agent as X.

A third aspect of the invention features a method of diagnosing disease in a mammal by administering the conjugate compound of the first aspect of the invention to the mammal (e.g., a human). In an embodiment, the conjugate compound includes a detectable label as X.

A fourth aspect of the invention features a method of making a conjugate compound of the first aspect of the invention by forming a disulfide bond between an agent described herein as being an X moiety (e.g., a cytotoxic, therapeutic, or diagnostic agent described herein) and an agent described herein as a Y moiety. In an embodiment, the Y moiety further includes, attached at its carboxy-terminal end, one or more of the Z targeting moieties described herein.

A fifth aspect of the invention features a kit that includes a conjugate

compound of the first aspect of the invention and instructions for administering it to a patient (e.g., a mammal, such as a human) for the treatment or detection of a disease. In other embodiments, the kit further includes one or more additional agents known to be administered to treat or detect the disease. The compositions of the invention may also include the cargo antisense molecule Genasense, which is a DNA-based treatment that targets Bcl-2, a protein expressed in high levels in cancer cells. The methods of the invention include the administration of this composition to treat malignant cancers (e.g., melanoma).

The compositions of the invention may also include the cargo antisense molecule that is the active componet of the therapeutic Vitravene (Isis

Pharmaceuticals, Carlsbad, California). The methods of the invention include the administration of this composition to treat cytomegalovirus infections in the eye, e.g., in patients with HIV.

The term "about" is used herein to mean a value that is ± 10% of the recited value.

By "detectable label" is meant any type of label which, when attached to a conjuage compound of the invention, renders the agent detectable. A detectable label may be toxic or non-toxic, and may have one or more of the following attributes, without restriction: fluorescence (Kiefer et al, WO 9740055), color, toxicity (e.g., radioactivity, e.g., a y-emitting radionuclide, Auger-emitting radionuclide, β-emitting radionuclide, an a-emitting radionuclide, or a positron-emitting radionuclide), radiosensitivity, or photosensitivity. Although a detectable label may be directly attached, for example, to an amino acid residue of the conjuage compound of the invention or via a disulfide linkage formed between a cysteine residue of the Y moiety and a thiol-containing group (e.g., a cysteine residue) of the X moiety), the detectable label may also be indirectly attached, for example, by being complexed with a chelating group that is attached (e.g., linked via a covalent bond or indirectly linked) to an amino acid residue or via a thiol linkage of the Y moiety of the conjuage compound of the invention. A detectable label may also be indirectly attached to an agent of the invention by the ability of the label to be specifically bound by a second molecule. One example of this type of an indirectly attached label is a biotin label that can be specifically bound by a second molecule, streptavidin. The second molecule may also be linked to a moiety that allows neutron capture (e.g., a boron cage as described in, for example, Kahl et al, Proc. Natl. Acad. Sci. USA

87:7265-7269, 1990).

A detectable label may also be a metal ion from heavy elements or rare earth ions, such as Gd³⁺, Fe³⁺, Mn³⁺, or Cr²⁺ (see, e.g., Curter, Invest. Radiol. 33(10):752- 761, 1998). Preferred radioactive detectable labels are radioactive iodine labels (e.g., ^,221, ¹²³1, ¹²⁴1, ¹²⁵I, or ¹³ ¾ that are capable of being coupled to each D- or L-Tyr or D- or L-4-amino-Phe residues present in the agents of the invention. Preferred nonradioactive detectable labels are the many known dyes that are capable of being coupled to NH₂-terminal amino acid residues.

Preferred examples of detectable labels that may be toxic to cells include ricin, diphtheria toxin, and radioactive detectable labels (e.g., ¹²²1, ^{1 3}1, ¹²⁴1, ¹²⁵1, ¹³¹I₅ ¹⁷⁷Lu, ⁶⁴Cu, ⁶⁷Cu, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Rc, ²¹¹ At, ²¹²Bi, ^{2 5}Ac, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ^n7mSn, ⁴⁷Sc, ¹⁰⁹Pd, ⁸⁹Sr, ¹⁵⁹Gd, ^,49Pm, ¹⁴²Pr, ⁱⁿAg, ¹⁶⁵Dy, ^2,3Bi, ^,nIn, ^114mIn, ²⁰¹Ti, ^195mPt, ¹⁹³Pt, ⁸⁶Y and ⁹⁰Y). These compounds, and others described herein may be directly or indirectly attached to an agent of the invention or its analogs. A toxic detectable label may also be a chemotherapeutic agent (e.g., camptothecins, homocamptothecins, 5-fluorouracil or adriamycin), or may be a radiosensitizmg agent (e.g., paclitaxel, gemcitabine, fluoropyrimidine, metronitozil, or the deoxycytidine analog 2',2' difluoro- 2'-deoxycytidine (dFdCyd) to which is directly or indirectly attached a conjuage compound of the present invention.

A detectable label, when coupled to a conjuage compound of the invention emits a signal that can be detected by a signal transducing machine. In some cases, the detectable label can emit a signal spontaneously, such as when the detectable label is a radionuclide. In other cases the detectable label emits a signal as a result of being stimulated by an external field such as when the detectable label is a relaxivity metal. Examples of signals include, without limitation, gamma rays, X-rays, visible light, infrared energy, and radio waves. Examples of signal transducing machines include, without limitation, gamma cameras including SPECT/CT devices, PET scanners, fluorimeters, and Magnetic Resonance Imaging (MRI) machines.

By "diagnostically effective amount" is meant a dose of detectably-labeled conjuage compound of the invention that, when administered internally to a mammal, is quantitatively sufficient to be detected by a signal transducing machine external to the mammal (e.g., a gamma camera used in gamma scintigraphy) but typically is quantitatively insufficient to produce a pharmacological effect.

By a "pharmaceutically acceptable excipient" is meant a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable excipient is physiological saline. Other physiologically acceptable excipients and their formulations are known to one skilled in the art and described, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed. A.R. Gennaro AR., 2000, Lippincott Williams & Wilkins).

By "polypeptide" or "peptide" is meant any chain of natural or unnatural amino acids, regardless of post-translational modification (e.g., glycosylation or

phosphorylation), constituting all or part of a naturally-occurring or non-naturally occurring polypeptide or peptide, as is described herein. Naturally occurring amino acids are any one of the following, alanine (A or Ala), cysteine (C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H, or His), isoleucine (I or He), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), ornithine (O or Orn), proline (P or Pro), hydroxyproline (Hyp), glutamine (Q or Gin), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), or tyrosine (Y or Tyr).

By "specifically binds" is meant that a conjugate compound of the invention

(e.g., the Z targeting moiety of the conjuage compound) recognizes and binds to a target cell, but does not substantially recognize and bind to a non-target, both in vivo and in a sample, e.g., an in vitro biological sample, that includes, e.g., target cells.

Preferably, the conjuage compounds of the invention bind target cells with at least 2, 5, 10, 20, 100, or 1000 fold greater affinity than they bind to non-target cells. For example, a target cell would include those cells that express a receptor that

specifically binds a receptor binding domain that is included in a conjugate compound of the invention as the Z targeting moiety, whereas non-target cells would lack the receptor, and thus would lack the ability to specifically bind such conjugate

compound. Alternatively, a target cell may include those cells that express a ligand that specifically binds a receptor that is included in a conjugate compound of the invention as the Z targeting moiety, whereas non-target cells would lack the ligand, and thus would lack the ability to specifically bind such conjugate compound.

By "substantially identical" is meant a protein, polypeptide, or nucleic acid exhibiting at least 75%, but preferably 85%, more preferably 90%, most preferably 95%, or even 99% or more (e.g., 100%) identity to a reference amino acid or nucleic acid sequence (e.g., any one of the proteins, polypeptides, or nucleic acids described herein). For polypeptides, the length of comparison sequences will generally be at least 10 amino acids, and preferably at least 20 amino acids, but may include the full- length amino acid sequence. For nucleic acids, the length of comparison sequences will generally be at least 30 nucleotides, preferably at least 60 nucleotides, and more preferably at least 120 nucleotides, but may include the full-length nucleic acid sequence.

Sequence identity is typically measured using BLAST^® (Basic Local

Alignment Search Tool) or BLAST^®2 with the default parameters specified therein (see, Altschul et al., J. Mol. Biol. 215:403-410, 1990); and Tatiana et al, FEMS Microbiol. Lett. 174:247-250, 1999). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. (A) Following trypsin "nicking" after Argininel94, native diphtheria toxin may be separated into an N-terminal -21 ,800 Da catalytic domain and the C-terminal -36,900 Da transmembrane and receptor binding domains. A partial N-terminal amino acid sequence of the transmembrane domain from Serine 195 to Asparagine235 which includes transmembrane helices 1 and 2 and the Tl motif is shown. The numbering for the diphtheria toxin transmembrane domain shown in Figure 1 is based on a recombinant form that includes an additional methionine residue at the amino-terminal end. This results in an increase of one amino acid relative to the native diphtheria toxin sequence. Accordingly, the cysteine at position 202 in Figure 1 (and Figure 5) corresponds to a cysteine at position 201 in the native diphtheria toxin sequence. (B) Effect of wild type DAB₃₈ IL-2 and variant site- directed mutants in transmembrane helix 1 on [¹⁴C]-leucine incorporation into HuT102 cells. DAB₃₈₉IL-2 (·); DAB(K213A) ₃₈₉IL-2; (Δ); DAB(K21 A) ₃₈₉IL-2, (O); DAB(K217A) ₃₈₉IL-2 (O); DAB(K222A) ₃₈₉IL-2 (□); (C) DAB₃₈₉IL2 (·); DAB(K213A,K215A)₃₈₉IL-2 (A); DAB(K215A,K217A) ₃₈₉IL2 (■); (D) DAB₃₈₉IL-2 (·); DAB(K213A,K215A,K217A,K222A)₃₈₉IL-2 (--□--). Data points (mean ± range) represent triplicate analysis at the indicated concentration from one representative experiment (n=3).

Figure 2. Effect of DAB₃₈₉IL-2 and the COPI binding domain swap mutant DAB(212p23)₃89lL-2 on [¹⁴C]-leucine incorporation into HuT102 cells. DAB₃₈₉IL-2 (·); DAB(212p23)3₈₉IL-2 (Δ). Data points (mean ± range) represent triplicate analysis at the indicated concentration from one representative experiment (n=3).

Figure 3. Autoradiographic analysis of GST and GST-DT140-217 to pull down reaction mixtures following in vitro protein synthesis of γι-COP, β'-COP, and ε-COP in rabbit reticulocyte lysates in the presence of [³⁵S]-methionine. Pull down reaction mixtures were dissolved in SDS-polyacrylamide gel loading buffer, electrophoresed on SDS-polyacrylamide gels, and following electrophoresis, gels were autoradiographed. Data are presented as the relative binding of [³⁵S]-labeled protein to each probe as measured by pixel density using Scion software. The data presented is plotted as mean± range of three independent experiments for each COPI subunit, respectively.

Figure 4. The aggregation and precipitation of COPI complex following exposure to the synthetic peptide DTB5 which carries the wild type sequence of transmembrane helix 1. Partially purified COPI enriched fractions from bovine liver (2.5 μg of total protein/ reaction) were incubated for one hr at room temperature with increasing concentrations of DTB5 (Table 2) in 40 \L of reaction buffer. The reaction mixture was then centrifuged (14,000 x g, 30 min) and the supernatant fluid and pellet fractions were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblot using both anti-yj-COP and anti-P-COP as probes. (A) Representative immunoblot analysis of COPI aggregation and precipitation following exposure to DTB5 (n = 4). (B) Aggregation and precipitation of COPI complex proteins by 2 mM DTB5 in the absence and presence of increasing concentrations of 1,3- cyclohexanebis(methylamine) (CBM) to the reaction mixture (n = 3). (C)

Aggregation and precipitation of COPI complex following exposure to the synthetic peptide wtp23 which carries the COPI complex binding sequence from the cytoplasmic tail of p23 adaptor protein. Immunoblot analysis was performed with only anti-P-COP antibodies (n = X).

Figure 5. Aggregation and precipitation of COPI complex following exposure to synthetic peptides which carry either wild type transmembrane helix 1 and helix 2 sequences (DTB5), variants which lack either the N- (KNOFF) or C- terminal (KCOFF) dilysine signatures, or the synthetic peptide in which all lysine have been replaced with alanine (KOFF). The assay was performed as described in Figure 4, except that following the electrophoresis of supernatant fluid and pellet fractions, the immunoblots were only probed with anti-p-COP antibodies.

Figure 6. In silico prediction of 25 amino acid fragment of transmembrane domain helices 1 and 2 (DWDVIRDKTKTKISSLKEHGH) of diphtheria toxin. The three dimensional model was constructed using the PEP-Fold algorithm (Maupetit et al, 2009) which is based on the structural alphabet SA letters to describe the conformation of four consecutive residues, and then couples the predicted series of SA letter to a greedy algorithm and course-grained force field. The resulting structure was the visualized using the Accelrys DS Visualizer (v 2.0.1.7347).

Figure 7. Schematic depiction of diphtheria toxin C-domain entry into a susceptible cell from the lumen of an early endosome. (1) diphtheria toxin binds to cell surface receptor and is (2) internalized through clathrin coated pits and vesicles into early endosomes. Upon acidification of the early endosomes (3) the

transmembrane domain of the toxin reorganizes to form a membrane pore through which the C-domain enters the cytosol (4). The facilitated entry of the C-domain is mediated by a Cytosolic Translocation Factor complex which includes β-COP, thioredoxin reductase and Hsp90. Once refolded into an active conformation, the C- domain catalyzes the ADP-ribosylation of elongation factor 2.

Figures 8A-C. (A) N-terminal amino acid sequence of anthrax lethal factor showing the relative positions of the Tl-like motif and the KXKXX COPI binding sequences that are essential for cytosolic delivery. (B) N-terminal amino acid sequence of the diphtheria toxin transmembrane domain showing the relative positions of the Tl motif and the KXKXX COPI binding sequences that are essential for catalytic domain entry. (C) Schematic diagram of the insertion of the diphtheria toxin catalytic and transmembrane domains into the transmembrane domain formed pore. As the Tl motif and KXKXX sequences emerge on the cytosolic surface of the endosomal vesicle they mimic cargo and adaptor sequences and present KXKXX binding sequences that are necessary for COPI binding and delivery of the toxin catalytic domains to the target cell cytosol. Red circles show the relative position of the KXKXX COPI binding signatures in the diphtheria toxin transmembrane domain and in anthrax lethal factor.

Figure 9. Schematic diagram of the effect of insertion of the mutant β-globin (IVS2-654) insert into the luc gene. Antisense PNA blockage of the 654 aberrant splice site results in correct splicing of intron-2 and translation of full length active luciferase.

Figure 10. Induction of luciferase activity in Luc-IVS2-654 CHO-K1 cells by 30 ng/mL antisense PNA-Lys₈ in the presence or absence of 300 ng/mL anthrax protective.

Figure 11. Schematic diagram of peptide-PNA - Fragment B 197 conjugate.

The construct carries antisense PNA sequences to block the aberrant 654 splice site in Luc-IVS2-654, the peptide sequences represent the C-terminal end of the diphtheria toxin catalytic domain with C sl86 that allow reconstitution with Fragment B197 through a disulfide bond. The construct is predicted to bind to the diphtheria hb-EGF- like precursor on the surface of Luc-IVS2-654 CHO-K1 cells and mediate

internalization into an endosomal vesicle. Following acidification, the Fragment B197 transmembrane will spontaneously insert into the vesicle membrane. The emergence of the Tl motif and KXKXX sequences and disulfide bond linked peptide- PNA sequences will bind COPI complex and result in the facilitated delivery and release of peptide-PNA into the cytosol. Delivery and release will be monitored by measuring the level of full length active luciferase that is expressed in target cells.

Figure 12. Total RNA was isolated from four independent Luc-IVS2-654 CHO Kl cell lines and the Luc-IVS2 control cell line using Trizol Reagent

(Invitrogen) and primers for sequences in luciferase reporter pGL3 (Promega) were used to amplify cDNA by PCR. The smaller properly processed Luc-IVS2 control is shown at the far right and gives rise to robust luciferase reporter activity in luciferase assays. In contrast the first four lanes from the left show both the appropriately spliced mature Luc-lVS2 luciferase encoding gene product (lower band) and the slower migrating Luc-IVS2-654 gene product which does not give rise to luciferase.

Figure 13. (A) Amino acid and nucleotide base sequence of peptide-siRNA that will be used to silence luc gene expression in CHO-Kl(luc+) cells. Peptide is covalently attached to the sense strand. (B) Amino acid and nucleotide base sequence of peptide-siRNA that will be used to silence luc gene expression in CHO-Kl(luc+) cells. Peptide is covalently attached to the anti-sense strand. Peptide component of the peptide-siRNA is shown in blue.

DETAILED DESCRIPTION

The Tl motif or the transmembrane helix 1 sequences of DT, anthrax LF and

EF, and botulinum neurotoxins serotypes A, C, and D on the cytoplasmic side of endosomal vesicle membranes can mimic cargo motifs and/or the KKXX and FFXX of adaptor proteins which are known to bind COPI complex proteins. I have discovered that at least three lysine residues within this region of the toxin(s) are essential for both cytotoxic activity in vivo and to mediate binding to COPI complex proteins in vitro. Further, replacement of the native transmembrane helix 1 with the 13 amino acid COPI binding domain from the cytoplasmic tail of p23 results in a domain swap mutant whose cytotoxic potency is identical to that of the wild type toxin. Thus, I have discovered that COPI complex binding to the N-terminal lysine- rich portion of the transmembrane domain of these toxins is an essential feature for C- domain delivery to the eukaryotic cell cytosol.

Once the lysine rich region of transmembrane helix 1 emerges from the trans- vesicle membrane pore it functions as a cargo and/or adaptor protein COPI binding site mimetic. Since the N-terminal portion of the DT transmembrane domain, with its disulfide bond linked C-domain, appears to pass unencumbered through the endosomal vesicle membrane pore, the protein-protein interaction(s) between these lysine residues and COPI complex appears to facilitate C-domain translocation and delivery to the cytosol much like pulling a string through a straw. The delivery of the DT C-domain into the target cell cytosol is facilitated by host cell factors which include Hsp90, thioredoxin reductase, and coatomer complex I (COPI). These interactions occur between a 10 amino acid motif, the Tl -motif, in transmembrane helix 1 of DT and adjacent KXKXX sequences and COPI complex proteins. The Tl motif and surrounding KXKXX sequences in the DT transmembrane helices 1 and 2 function as a mimetic(s) to the COPI binding regions found in the cytoplasmic tail of p23/24 adaptor proteins. The COPI coatomer complex plays an essential role in the efficient delivery of toxins, such as DT and other toxins, e.g., anthrax LF and EF and botulinum neurotoxins serotypes A, C, and D, across the endosomal vesicle membrane and into the eukaryotic cell cytosol. The requirements for COPI complex protein for both the DT C-domain and LF entry into the cytosol confirm that there is a common mechanism of entry for these highly divergent bacterial protein toxins.

The present invention features the use of one or more KXKXX COP-I binding motifs, such as those found within the transmembrane domain of toxins, such as DT, anthrax LF and EF, and botulinum neurotoxins serotypes A, C, and D, to produce conjugate compounds that can be used to deliver therapeutic and/or diagnostic agents to cells for the treatment of diseases or disorders. The conjugate compounds utilize these regions of, e.g., Diphtheria and anthrax toxins that have evolved as highly efficient nano-machines that function to deliver their respective catalytic domain "cargo" to the eukaryotic cell cytosol. Now that the overall process and structural requirements necessary for "cargo" delivery are more clearly defined, this machinery can be used for cell specific delivery of cargo, such as siRNA, dsRNA, RNAi, protein nucleic acids (PNA), and protein agents (e.g., transcription factors and other polypeptides) by "cargo" domain substitution using a toxin-based platform. In other words, the C-domain of toxins, such as, e.g., DT, anthrax LF and EF, and botulinum neurotoxins serotypes A, C, and D, can be removed and replaced with siRNA, dsRNA, RNAi, protein nucleic acids (PNA), and protein agents to the cytosol of a target cell.

A conjugate compound of the invention has the general formula:

X-Y-Z (Formula 1)

wherein X is selected from a cytotoxic agent (e.g., those agents described herein and generally known in the art), a therapeutic agent (e.g., a siRNA, dsRNA, RNAi, a protein nucleic acid (PNA) molecule, and a protein agent (e.g., a transcription factors and other polypeptides)), or a detectable label having at least one thiol-containing group (e.g., a cysteine residue or other thiol forming group) for the formation of a disulfide bond with the Y moiety (e.g., in the case of a nucleic acid molecule the thiol group may be located at either the 3' or 5' end of X or at any position within the nucleic acid molecule; in the case of a polypeptide the thiol group may be located at either the amino- or carboxy-terminal end or at any position within the polypeptide sequence);

Y is a polypeptide containing a lysine-rich domain that is capable of interacting with cellular COPI complex proteins and that includes at least one bond that is cleavable by an intracytosolic enzyme (e.g., the polypeptide may include a cysteine residue that forms a disulfide bond with the at least one thiol group (e.g., a cysteine residue) of the X moiety that is cleavable by an intracytosolic enzyme (e.g., thioredoxin reductase)), in which the lysine-rich domain includes one or more lysine rich motifs having a dibasic signature (e.g., KKXX, XKXX) and/or an aromatic amino acid sequences (e.g,. FFXXBB(X)„,; e.g., the lysine rich motif can be selected from the transmembrane helix 1 of DT, the Tl motif, or an amino acid sequence having at least 80% or more (e.g., 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to a contiguous amino acid sequence corresponding to at least amino acids 201 to 222 of DT (e.g., the lysine rich region has substantial sequence identity (80% or more, as described above) to a contiguous amino acid sequence corresponding to at least amino acids 201-235, amino acids 1 5 to 222, amino acids 195 to 235, amino acids 201 to 300, amino acids 195 to 300, amino acids 201 to 389, or amino acids 195 to 389, of DT); and

Z is a polypeptide targeting moiety (e.g., a receptor binding domain) that is bound to Y at its carboxy-terminal end.

The X moiety may also include a linker that connects the therapeutic agent (e.g., a siRNA, dsRNA, RNAi, a protein nucleic acid (PNA) molecule, or a protein agent (e.g., a transcription factors and other polypeptides)) to a thiol-containing group that can be used to form a disulfide bond with the thiol-containing group of the Y moiety.

In an embodiment, the transmembrane and receptor binding domains of the non- toxic DT mutant CRM 197 and the DT-related fusion protein toxins are used as structural platforms for the development of non-viral, cell receptor-specific siRNA cytosolic delivery systems. These systems employ their respective cell receptor- specific targeting and endosomal vesicle membrane pore forming ability and COPI binding motifs for enhanced delivery of peptide-siR As to the target cell cytosol.

Therapeutic or Diagnostic Agents for Use in the Conjugate Compounds of the Invention

Conjugate compounds of the invention can include therapeutic or diagnostic agents as the "cargo" moiety (i.e., the X moiety according to Formula I above). The "cargo" replaces the C-domain within the toxin platform and is attached to the Y moiety, which corresponds to the transmembrane and receptor binding domain of the toxin platform, via a disulfide linkage. The "cargo" can be modified to include a thiol-linkage that allows the formation of this disulfide linkage. The addition of a thiol-modification to a nucleic acid molecule, a polypeptide or protein, or a chemical compound is well-known in the art (see, e.g., United States Patent Application Publication No. 2005/0118099, incorporated herein by reference). For example, the X moiety is prepared so that it is suitable for thiol-specific attachment via a free cysteine to the Y moiety of the conjugate compound. Thiol-specific drug attachment to a peptide analog can be direct or indirect, i.e. via a chelator (e.g., MX-DTPA, which is useful in preparing the peptide analogs of the invention; the maleimido derivatives of MX-DTPA chelator is reactive with thiol groups of a peptide portion of the X moeity (i.e., SH groups of one or more free cysteines) to form a thioether linkage). The thiol attachment methods of the present invention are generally applicable to the attachment of drugs/chelators to the Y/Z portion of the conjugate compound. The thiol linkage can be a stable linkage, for example as a thioether linkage. Thus, in one embodiment of the invention, a drug or chelator is functionalized with a thiol reactive group (e.g., a maleimido group) that provides a stable thioether linkage. Optionally, a drug can comprise a cleavable site, such that the X moiety can be released from Y moiety (e.g., by reducing the disulfide bond). Here, the thiol linkage can be labile, for example, the X moiety is functionalized with a thiol group enabling formation of a disulfide bond with the Y moiety. A conjugate so prepared is redox active, such that it is stable in the serum and is released upon entry into the reducing environment of the cell cytosol. Alternatively, other types of linkages are envisioned, e.g.,

representative cleavable sites include acid-labile and enzyme-labile sites.

Therapeutic Agents as the X Moiety

Therapeutic agents that can be used as the "cargo" of the compounds of the invention include cytotoxic polypeptides, such as cytochrome c, caspase 1-10, granzyme A or B, tumor necrosis factor-alpha (TNF-a), TNF-β, Fas, Fas ligand, Fas-associated death doman-like IL-Ι β converting enzyme (FLICE), TRAIL/AP02L, TWEAK/AP03L, Bax, Bid, Bik, Bad, Bak, RICK, vascular apoptosis inducing proteins 1 and 2 (VAP1 and VAP2), pierisin, apoptosis-inducing protein (AIP), IL-l propiece polypeptide, apoptin, apoptin-associated protein 1 (AAP-1), endostatin, angiostatin, and biologically-active fragments thereof. The cargo (X) moiety can also be one or more therapeutic agents such as cyclophosphamide, camptothecin, homocamptothecin, colchicine, combrestatin, combrestatin, rhizoxin, dolistatin, ansamitocin p3, maytansinoid, auristatin, caleachimicin, methotrexate, 5-fluorouracil (5-FU), doxorubicin, paclitaxel, docetaxel, cisplatin, carboplatin, tamoxifen, raloxifene, letrozole, epirubicin, bevacizumab, pertuzumab, trastuzumab, and derivatives thereof.

The X moiety can also be selected from nucleic acid molecules, e.g., siR As, dsRNAs, and other nucleic acid molecules that are known in the art to silence gene expression. In an embodiment, the nucleic acid molecules are those that silence genes that express polypeptides that are known to be involved in disease.

siRNA molecules for use in the treatment of diseases are known in the art (see, e.g., U.S. Patent Nos. 7,056,704; 7,678,896; 7,678,897; and 7691998; and U.S. Patent Application Publication Nos. 20100062051 (entitled Composition for Treatment of Cervix Cancer); 20100062436; 20100062951; 20100062967; 20100063131;

20100063132 (entitled Small Interfering RNA and Pharmaceutical Composition for Treatment of Hepatitis B Comprising the Same); 20100063134 (entitled Treatment of Neurodegenerative Disease Through Intracranial Delivery of siRNA); and 20100063308; 20080249046; 20080260854; 20090318536; and 20100098664; each of which is incorporated by reference herein in their entirety). Any of the siRNA molecules described in these publications can be used as the X moiety in the conjugate compounds of the invention for use in the treatment of diseases for which the siRNA molecules are known to treat or were developed to treat.

The X moiety may also be a transcription factor or a nucleic acid molecule that encodes a transcription factor. The transcription factor can be selected from helix-turn- helix motif proteins, homeodomain proteins, zinc finger motif proteins, steroid receptor proteins, leucine zipper motif proteins, helix-loop-helix motif proteins, and β-sheet motif proteins. In other embodiments, the X moiety is a nucleic acid binding compound that binds nonspecifically to nucleic acids and is selected from the group consisting of poly- L-lysine, protamine, histone and spermine. In a preferred embodiment, the X moiety is a nucleic acid binding domain that binds the coding region of a ribosome inactivating protein such as saporin. Transcription factors for use as the X moiety in the preparation of conjugate compounds of the invention are known in the art (see, e.g., U.S. Patent No. 6,037,329, incorporated by reference herein in its entirety).

The X moiety can also be a cytokine or a nucleic acid molecule that encodes a cytokine. For example, the X moiety can be, or can encode, a cytokine such as GM- CSF, IL-2, IL-12, IL-13, or IL-5. The gene that encodes a cytokine can be used alone or together with one or more foreign antigen genes to produce, e.g., a vaccine that results in enhanced immune responses, or to modulate immunity directed more specifically towards cellular, humoral, or mucosal responses.

The X moiety may also be a growth factor polypeptide or a nucleic acid molecule that encodes the growth factor selected from leptin receptor (LPTR), granulocyte colony stimulating factor receptor (GCSFR), LIF/OSM/CNTF common beta chain (GPI30), leukemia inhibiting factor receptor (LIFR), oncostatin-M receptor beta chain (OSMR), interleukin-12 receptor beta-1 chain (IL12RB1), and interleukin-12 receptor beta-2 chain (IL 12RB2). These receptor chains homodimerize (GCSFR, GPI30, LPTR) or

heterodimerize (GPI30 with LIFR or OSMR, IL12RB1 with IL12RB2) such as G-CSF, GM-CSF or M-CSF), SCF (stem cell factor), SCPF (stem cell proliferation factor), various Interleukins (IL1 , IL4, IL5, IL6, IL11, IL12), LIF, TGF-β, MIP-1-α, TNF-a, and also many other low molecular weight factors. The X moiety may also be a growth factor polypeptide or a nucleic acid molecule that encodes the growth factor (e.g., a growth factor selected from stem cell factor (SCF), FLT3, IL-3, IL-6, GSF, GM-CSF, and erythropoietin). In an embodiment, the conjugate compound can include a targeting moiety (Z) that targets the conjugate compound to a stem cell (e.g., a hemotopoeitic stem cell or a mesenchymal stem cell) and can include an X moiety, such as one of the growth factors described above, that promotes the differentiation of stem cells into a desired lineage (e.g., neuronal, hepatic, osteogenic, chondrogenic, tendonogenic, ligamentogenic, myogenic, marrow stromagenic, adipogenic or dermogenic lineage). Growth factors that stimulate the differentiation of stem cells are known in the art (see, e.g,. U.S. Patent No. 5,942,225, incorporated by reference herein).

In an embodiment, the conjugate compound can be used to induce stem cell differentiation into neural stem cells or neural cells in vivo, ex vivo, or in vitro. If done ex vivo or in vivo, these cells can then be transplanted to an injured site to treat a

neurological condition, e.g., Alzheimer's disease, Parkinson's disease, multiple sclerosis, cerebral infarction, spinal cord injury, or other central nervous system disorder, see, e.g., Morizane et al., (2008), Cell Tissue Res., 331(l):323-326; Coutts and Keirstead (2008), Exp. Neurol., 209(2):368-377; Goswami and Rao (2007), Drugs, 10(10):713-719.

For the treatment of Parkinson's disease, the stem cells may be differentiated into dopamine-acting neurons using a conjugate compound of the invention and then transplanted into the striate body of a subject with Parkinson's disease. For the treatment of multiple sclerosis, neural stem cells may be differentiated into oligodendrocytes or progenitors of oligodendrocytes using a conjugate compound of the invention and then transferred to a subject suffering from MS.

For the treatment of any neurologic disease or disorder, a successful approach may be to introduce neural stem cells differentiated using a conjugate compound of the invention to the subject. For example, in order to treat Alzheimer's disease, cerebral infarction or a spinal injury, the stem cells may be differentiated into neural stem cells using a conjugate compound of the invention followed by transplantation of the cells into the injured site. The induced cells may also be engineered to respond to cues that can target their migration into lesions for brain and spinal cord repair, e.g., Chen et al., (2007), Stem Cell Rev., 3(4):280-288.

Diseases other then neurological disorders may also be treated by stem cell therapy using cells from induced cells, e.g., induced multipotent or pluripotent stem cells and differentiated using a conjugate compound of the invention. Degenerative heart diseases such as ischemic cardiomyopathy, conduction disease, and congenital defects could benefit from this type of stem cell therapy, see, e.g. Janssens et al., (2006), Lancet, 367:1 13-121.

Stem cells may also be differentiated using a conjugate compound of the invention into pancreatic islet cells (or primary cells of the islets of Langerhans), which may then be transplanted into a subject suffering from diabetes (e.g., diabetes mellitus, type 1), see e.g., Burns et al., (2006) Curr. Stem Cell Res. Ther., 2:255-266. In some embodiments, pancreatic beta cells derived from induced cells and differentiated using a conjugate compound of the invention may be transplanted into a subject suffering from diabetes (e.g., diabetes mellitus, type 1).

In other examples, hepatic cells or hepatic stem cells can be differentiated using a conjugate compound of the invention and transplanted into a subject suffering from a liver disease, e.g., hepatitis, cirrhosis, or liver failure.

Conjugate compounds of the invention may also include a nucleic acid molecule, PNA molecule, or polypeptide as the X moiety that is an anti-viral (e.g., an anti- retro viral, for use in the treatment of HIV infection). Conjugate compound of the invention can also include an interferon (IFN), such as IFN-a, as the X moiety. In preferred embodiments, the conjugate compound includes, as the X moiety, a nucleic acid molecule (e.g., a siRNA, dsRNA, or PNA molecule) or a polypeptide that is an antiviral agent for use in the treatment of a viral infection. Preferably, the conjugate compound includes an X moiety that can be used as an anti-viral in the treatment of a virus selected from a member of the Flaviviridae family (e.g., a member of the

Flavivirus, Peslivirus, and Hepacivirus genera), which includes the hepatitis C virus, Yellow fever virus, Tick-borne viruses, Japanese encephalitis virus, West Nile virus, and yellow fever virus; a member of the Arenaviridae family, which includes the Lassa virus; a member of the Bunyaviridae family (e.g., a member of the Hantavirus, Nairovirus, Orthobunyavirus, and Phlebovirus genera); a member of the Filoviridae family, which includes the Ebola virus and the Marburg virus; a member of the Togaviridae family (e.g., a member of the Alphavirus genus), which includes the Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Sindbis virus, rubella virus, and Semliki Forest virus; a member of the Poxviridae family (e.g., a member of the Orthopoxvirus genus), which includes the smallpox virus, monkeypox virus, and vaccinia virus; a member of the Herpesviridae family, which includes the herpes simplex virus (HSV; types 1, 2, and 6), human herpes virus (e.g., types 7 and 8), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Varicella-Zoster virus, and Kaposi's sarcoma associated-herpesvirus (KSHV); a member of the Orthomyxoviridae family, which includes the influenza virus (A, B, and C), such as the H5N1 avian influenza virus or H1N1 swine flu; a member of the

Coronaviridae family, which includes the severe acute respiratory syndrome (SARS) virus; a member of the Rhabdoviridae family, which includes the rabies virus and vesicular stomatitis virus (VSV); a member of the Paramyxoviridae family, which includes the human respiratory syncytial virus (RSV), Newcastle disease virus, hendravirus, nipahvirus, measles virus, rindeipest virus, canine distemper virus, Sendai virus, human parainfluenza virus (e.g., 1, 2, 3, and 4), rhinovirus, and mumps virus; a member of the Picornaviridae family, which includes the poliovirus, human enterovirus (A, B, C, and D), hepatitis A virus, and the coxsackievirus; a member of the

Hepadnaviridae family, which includes the hepatitis B virus; a member of the

Papillamoviridae family, which includes the human papilloma virus; a member of the Parvoviridae family, which includes the adeno-associated virus; a member of the Astroviridae family, which includes the astrovirus; a member of the Polyomaviridae family, which includes the JC virus, BK virus, and SV40 virus; a member of the Calciviridae family, which includes the Norwalk virus; a member of the Reoviridae family, which includes the rotavirus; and a member of the Retroviridae family, which includes the human immunodeficiency virus (HIV; e.g., types 1 and 2), and human T- lymphotropic virus Types I and II (HTLV-1 and HTLV-2, respectively).

The X moiety of the invention may also be a cytotoxic agent. Examples include, e.g., antineoplastic agents such as: Acivicin; Aclarubicin; Acodazole Hydrochloride;

Acronine; Adozelesin; Adriamycin; Aldesleukin; Altretamine; Ambomycin; A.

metantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin;

Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin

Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone;

Camptothecin; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin

Hydrochloride; Carzelesin; Cedefmgol; Chlorambucil; Cirolemycin; Cisplatin;

Cladribine; Combretestatin A-4; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; DACA (N- [2- (Dimethyl-amino) ethyl] acridine-4-carboxamide);

Dactinomycin; Daunorubicin Hydrochloride; Daunomycin; Decitabine; Dexormaplatin;

Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Dolasatins; Doxorubicin;

Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone

Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Ellipticine;

Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride;

Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium;

Etanidazole; Ethiodized Oil 1 131 ; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole

Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate;

Fluorouracil; 5-FdUMP; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198; Homocamptothecin; Hydroxyurea; Idarubicin

Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon

Alfa-nl; Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b; Iproplatin;

Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole

Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride;

Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate;

Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate;

Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin;

Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin;Ormaplatin; Oxisuran; Paclitaxel;

Pegaspargase; Peliomycin; Pentamustine; PeploycinSulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Rhizoxin; Rhizoxin D; Riboprine; Rogletimide; Safmgol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfm; Teniposide; Teroxirone;

Testolactone; Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin; Tirapazamine; Tomudex; TOP53; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine;

Vinblastine Sulfate; Vincristine; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride; 2-Chlorodeoxyadenosinc; 2' Deoxyformycin; 9-aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid; 2chloro-2'-arabino-fluoro-2'- deoxyadenosine; 2-chloro-2'-deoxyadenosine; anisomycin; trichostatin A; hPRL-G129R; CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlor ethamine);

cyclophosphamide; nielphaian; chlorambucil; ifosfamide; busulfan; N-methyl- Nnitrosourea (MNU); N, N'-Bis (2-chloroethyl)-N-nitrosourea (BCNU); N- (2- chloroethyl)-N' cyclohexyl-N-nitrosourea (CCNU); N- (2-chloroethyl)-N - (trans-4- methylcyclohexyl-N-nitrosourea (MeCCNU); N- (2-chloroethyl)-N'- (diethyl) ethylphosphonate-N-nitrosourea (fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide; temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; Cisplatin;

Carboplatin; Ormaplatin; Oxaliplatin;C 1-973; DWA 2114R; JM216; JM335; Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine; 6-Mercaptopurine; 6- Thioguanine; Hypoxanthine; teniposide 9-amino camptothecin; Topotecan; CPT- 11 ; Doxorubicin; Daunomycin; Epirubicin; darubicin; mitoxantrone; losoxantrone;

Dactinomycin (Actinomycin D); amsacrine; pyrazoloacridine; all-trans retinol; 14- hydroxy-retro-retinol; all-trans retinoic acid; N- (4- Hydroxyphenyl) retinamide; 13-cis retinoic acid; 3-Methyl TTNEB; 9-cis retinoic acid; fludarabine (2-F-ara-AMP); or 2- chlorodeoxyadenosine (2-Cda).

Other therapeutic compounds that can be used as the X moiety include, but are not limited to, 20-pi-l,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;

aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;

antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein- 1 ; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine;

atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;

azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta- alethine; betaclamycin B; betulinic acid: bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bleomycin A2; bleomycin B2; bropirimine; budotitane; buthionine sulfoximine; calcipotriol;

calphostin C; camptothecin derivatives (e.g., 10-hydroxy-camptothecin); canarypox

IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A

; collismycin B; combretastatin A4; combretastatin analogue; conagenin;

crambescidin 816 ; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanihraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; 2'deoxycoformycin

(DCF); deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin

B; didox; diethylnorsperrnine; dihydro-5-azacytidine; dihydrotaxol, 9- ; dioxamycin; diphenyl spiromustine; discodermolide; docosanol; dolasetron; doxifluridine;

droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;

edrecolomab; eflornithinc; clemene; emitefur; epirubicin; epothilones (A, R = H; B, R

= Me); epithilones; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide; etoposide 4'-phosphate (etopofos); exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fiuasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine;

ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;

heregulin; hexamethylene bisacetamide; homoharringtonine (HHT); hypericin;

ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;

imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane;

iododoxorubicin; ipomeanol, 4- ; irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin: letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maytansine; mannostatin A; marimastat;

masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors;

menogaril; rnerbarone; meterelin; methioninase; metoclopramide; M1F inhibitor; ifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mithracin; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A + myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 -based therapy; mustard anticancer agent; mycaperoxide B;

mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase;

nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06- benzyl guanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin;

oxaunomycin; paclitaxel analogues; paclitaxel derivatives; palauamine;

palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin;

pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;

phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride;

pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor;

platinum complex; platinum compounds; platinum-triamine complex;

podophyllotoxin; porfimer sodium; porfiromycin; propyl bis-acridone: prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B 1 ; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1 ; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein;

sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol;

somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1 ; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic

glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;

temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine;

thalidomide; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;

tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. The X moiety may also be a lytic peptide. Such lytic peptides induce cell death and include, but are not limited to, streptolysin O; stoichactis toxin; phallolysin; staphylococcus alpha toxin; holothurin A; digitonin; melittin; lysolecithin;

cardiotoxin; and cerebratulus A toxin (Kem et al, J. Biol. Chem. 253(16):5752-5757, 1978). The X moiety may also be a synthetic peptide that shares some sequence homology or chemical characteristics with any of the naturally occurring peptide lysins; such characteristics include, but are not limited to, linearity, positive charge, amphipathicity, and formation of alpha-helical structures in a hydrophobic environment (Leuschner et al, Biology of Reproduction 73:860-865, 2005). Agents of the invention can also be coupled to an agent that induces complement-mediated cell lysis such as, for example, the immunoglobulin F_c subunit. The X moiety may also be selected from any member of the phospholipase family of enzymes (including phospholipase A, phospholipase B, phospholipase C, or phospholipase D) or to a catalytically-active subunit thereof.

The X moiety can also include a radioactive agent, including, but not limited to: Fibrinogen ¹²⁵I; Fludeoxyglucose ¹⁸F; Fluorodopa ¹⁸F; Insulin ^l25I; Insulin ¹³¹I; lobenguane ^I23I; Iodipamide Sodium ^,31I; Iodoantipyrine ¹³¹I; Iodocholesterol ¹³¹I ; lodohippurate Sodium ¹²³I; Iodohippurate Sodium ^{1 5}ϊ; Iodohippurate Sodium ¹³¹I;

Iodopyracet 125 I; Iodopyracet 131 I; lofetamine Hydrochloride 12 I; Iomethin 125 I;

Iomethin ¹³¹I; Iothalamate Sodium ¹²⁵I; Iothalamate Sodium ¹³¹I; tyrosine ¹³¹I;

Liothyronine ¹²⁵I; Liothyronine ¹³¹I; Merisoprol Acetate ¹⁹⁷Hg; Merisoprol Acetate ²⁰³Hg; Merisoprol ¹⁹⁷Hg; Selenomethionine ⁷⁵Se; Technetium ^99mTc Antimony Trisulfide Colloid; Technetium ^99mTc Bicisate; Technetium ^99mTc Disofenin;

Technetium ^99mTc Etidronate; Technetium ^99mTc Exametazime; Teclinetium ^99mTc Furifosmin; Technetium ⁹⁹ⁿⁱTc Gluceptate; Technetium ^99mTc Lidofenin; Technetium ^99mTc Mebrofenin; Technetium ^99mTc Medronate; Technetium ^99mTc Medronate Disodium; Technetium ^99mTc Mertiatide; Technetium ^99mTc Oxidronate; Technetium ^99mTc Pentetate; Technetium ^99mTc Pentetate Calcium Trisodium; Technetium ^{9 ni}Tc Sestamibi; Technetium ^99mTc Siboroxime; Technetium ^99mTc; Succimer; Technetium ^99mTc Sulfur Colloid; Technetium ^99mTc Teboroxime; Technetium ^99mTc Tetrofosmin; Technetium ^99mTc Tiatide; Thyroxine ^{1 5}I; Thyroxine ^{I 31}I; Tolpovidone ¹³¹I; Triolein ¹²⁵1; or Triolein ¹³¹I. The X moiety can also include, for example, anti-cancer Supplementary

Potentiating Agents, including, but not limited to: Tricyclic anti-depressant drugs (e.g., imipramine, desipramine, amitryptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapinc, and maprotiline); non-tricyclic anti-depressant drugs (e.g., sertraline, trazodone, and citalopram); Ca²⁺ antagonists (e.g., verapamil, nifedipine, nitrendipine, and caroverine); Calmodulin inhibitors (e.g., prenylamine, trifluoroperazine, and clomipramine); Amphotericin B; Triparanol analogs (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine); antihypertensive drugs (e.g., reserpine); Thiol depleters (e.g., buthionine and sulfoximine) and Multiple Drug

Resistance reducing agents such as Cremaphor EL.

The X moiety can also be selected from antimetabolic agents, such as methotrexate. Antimetabolites for use as the X moiety include, but are not limited to, the following compounds and their derivatives: azathioprine, cladribine, cytarabine, dacarbazine, fludarabine phosphate, fluorouracil, gencitabine chlorhydrate,

mercaptopurine, methotrexate, mitobronitol, mitotane, proguanil chlorohydrate, pyrimethamine, raltitrexed, trimetrexate glucuronate, urethane, vinblastine sulfate, vincristine sulfate, etc. More preferably, the any of the agents of the invention can be coupled to a folic acid-type antimetabolite, a class of agents that includes, for example, methotrexate, proguanil chlorhydrate, pyrimethanime, trimethoprime, or trimetrexate glucuronate, or derivatives of these compounds.

The X moiety can also be selected from a member of the anthracycline family of neoplastic agents, including but not limited to aclarubicine chlorhydrate,

daunorubicine chlorhydrate, doxorubicine chlorhydrate, epirubicine chlorhydrate, idarubicine chlorhydrate, pirarubicine, or zorubicine chlorhydrate; a camptothecin, or its derivatives or related compounds, such as 10, 11 methylenedioxycamptothecin; or a member of the maytansinoid family of compounds, which includes a variety of structurally-related compounds, e.g., ansamitocin P3, maytansine, 2'-N- demethylmaytanbutine, and maytanbicyclinol.

The X Moiety can also be a Detectable Label

Detectable labels can be used as the X moiety to prepare conjugate compounds of the invention for use as diagnostic agents. In this embodiment, a detectable label is used as the "cargo" of the compounds of the invention. Detectable labels can be selected from a radioactive, bioluminescent, fluorescent, or heavy metal label, or an epitope tag.

Detectable labels of the conjugate compounds can include radioactive metals for use in radiographic imaging or radiotherapy. Preferred radioisotopes also include ^99mTc, ^{5 I}Cr, ⁶⁷Ga, ⁶⁸Ga, ^mIn, ¹⁶⁸Yb, ^14CLa, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁵⁶Ho, ¹⁶⁵Dy, ⁶⁴Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²ⁿBi, ²¹²Bi, ^2I3Bi, and ²¹⁴Bi. The choice of metal is determined based on the desired therapeutic or diagnostic application.

The metal complexes of the invention are useful as diagnostic and/or

therapeutic agents. A detectable label may be a metal ion from heavy elements or rare earth ions, such as Gd , Fe , Mn , or Cr . Conjugates that include paramagnetic or superparamagnetic metals are useful as diagnostic agents in MRI imaging

applications. Paramagnetic metals that may be used in the conjugates include, but are not limited to, chromium (III), manganese (II), iron (II), iron (III), cobalt (II), nickel

(II) , copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium

(III) , terbium (III), dysprosium (III), holmium (III), erbium (III), and ytterbium (III). Preferably, the conjugate compound has a relaxtivity of at least 10, 12, 15, or 20 mM^"1 sec^"1 wherein Z is the concentration of paramagnetic metal.

Fluorescent molecules that can also serve as detectable labels include green fluorescent protein (GFP), enhanced GFP (eGFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), and dsRed. In an embodiment, the bioluminescent molecule is luciferase. In another embodiment, the epitope tag is c- myc, hemagglutinin, or a histidine tag.

Polypeptides for Use as the Y Moeity in the Compositions of the Invention

Y is a polypeptide containing a lysine-rich domain that is capable of

interacting with cellular COPI complex proteins and that includes at least one bond that is cleavable by a cytosolic enzyme (e.g., the polypeptide may include a cysteine residue that forms a disulfide bond with the at least one thiol group (e.g., a cysteine residue) of the X moiety that is cleavable by an intracytosolic enzyme (e.g.,

thioredoxin reductase)). The lysine-rich domain can include one or more lysine rich motifs having a dibasic signature (e.g., XX and KXKXX) and/or an aromatic amino acid sequences (e.g,. FFXXBB(X),,,. For example, the lysine rich motif can be selected from the transmembrane helix 1 of DT, the Tl motif, or an amino acid sequence having at least 80% or more (e.g., 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to a contiguous amino acid sequence corresponding to at least amino acids 201 to 222 of DT (e.g., the lysine rich region has substantial sequence identity (80% or more, as described above) to a contiguous amino acid sequence corresponding to at least amino acids 201-235, amino acids 195 to 222, amino acids 195 to 235, amino acids 201 to 300, amino acids 195 to 300, amino acids 201 to 389, or amino acids 195 to 389, of DT). Other amino acid sequences for use as the Y moiety can be selected from the transmembrane domains of other toxins (see, e.g., Table 1). These sequences can be modified at their amino-terminal end to include a cysteine residue in order to establish the formation of a disulfide bond between the Y/Z moieties and the X moiety of the conjugate compound.

Table 1. Polypeptide sequences for use as the Y moiety

Database accession numbers are given in parentheses. Other polypeptides for use as the Y moiety include the consensus peptide sequence of CTF-binding moiety: RDKTKTKIESLKEHGPIKNS, the consensus peptide sequence of CTF-binding moiety including KXKXX sequences in bold:

KTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQY, and amino acids 201-256 of the CTF binding moiety:

C₂₀₁INLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTA LE₂₅₆.

Other polypeptides for use as the Y moiety are described in, e.g., US 2008- 0306003, incorporated by reference herein in its entirety, and below.

Targeting Moieties (Z) for Use in the Compositions of the Invention

The compounds of the invention can be targeted to a specific cell or cells by using a targeting moiety (Z) that directs the compounds to a desired target cell. The Z targeting moiety is selected based on its ability to target conjugate compounds of the invention to a desired or selected cell population that expresses the corresponding binding partner (e.g., either the corresponding receptor or ligand) for the selected Z targeting moiety. For example, a conjugate compound of the invention could be targeted to cells expressing epidermal growth factor receptor (EGFR) by selected epidermal growth factor (EGF) as the Z targeting moiety.

If desired, one or more of the Z targeting moieties described herein can also be used as the X moiety. In an embodiment, the targeting moiety (Z) is a receptor binding domain. In other embodiments, the targeting moiety (Z) is or specifically binds to a protein selected from the group consisting of insulin, insulin-like growth factor receptor 1 (IGF1R), IGF2R, insulin-like growth factor (IGF; e.g., IGF 1 or 2), mesenchymal epithelial transition factor receptor (c-met; also known as hepatocyte growth factor receptor (HGFR)), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), heregulin, fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), platelet-derived growth factor (PDGF), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor (VEGF), tumor necrosis factor receptor (TNFR), tumor necrosis factor alpha (TNF-a), TNF-β, folate receptor (FOLR), folate, transferring, transferrin receptor (TfR), mesothelin, Fc receptor, c-kit receptor, c-kit, an integrin (e.g., an a4 integrin or a β-l integrin), P-selectin, sphingosine-1 -phosphate receptor- 1 (SI PR), hyaluronate receptor, leukocyte function antigen-1 (LFA-1), CD4, CDl l, CD18, CD20, CD25, CD27, CD52, CD70, CD80, CD85, CD95 (Fas receptor), CD106 (vascular cell adhesion molecule 1 (VCAM1), CD 166 (activated leukocyte cell adhesion molecule (ALCAM)), CD178 (Fas ligand), CD253 (TNF-related apoptosis-inducing ligand (TRAIL)), ICOS ligand, CCR2, CXCR3, CCR5, CXCL12 (stromal cell-derived factor 1 (SDF- 1 )), interleukin 1 (IL-1), IL-lra, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, CTLA-4, MART-1, gplOO, MAGE-1, ephrin (Eph) receptor, mucosal addressin cell adhesion molecule 1

(MAdCAM-1), carcinoembryonic antigen (CEA), Lewis^Y, MUC-1, epithelial cell adhesion molecule (EpCAM), cancer antigen 125 (CA125), prostate specific membrane antigen (PSMA), TAG-72 antigen, and fragments thereof. In a further embodiment, the targeting moiety, Z, is erythroblastic leukemia viral oncogene homolog (ErbB) receptor (e.g., ErbBl receptor; ErbB2 receptor; ErbB3 receptor; and ErbB4 receptor).

The Z targeting moiety can also be selected from bombesin, gastrin, gastrin- releasing peptide, tumor growth factors (TGF), such as TGF-a and TGF-β, and vaccinia virus growth factor (VVGF). Non-peptidyl ligands can also be used as the Z targeting moiety and may include, for example, steroids, carbohydrates, vitamins, and lectins. The Z targeting moiety may also be selected from a peptide, such as somatostatin (e.g., a somatostatin having the core sequence cyclo[Cys-Phe-D-Trp-Lys-Thr-Cys], and in which, preferably, the C-terminus of the somatostatin analog is: Thr-NFL), a somatostatin analog (e.g., octreotide and lanreotide), bombesin, a bombesin analog, or an antibody, such as a monoclonal antibody.

Other peptides for use as the Z targeting moiety in the conjugate compounds of the invention can be selected from iSS peptides and analogs, urotensin II peptides and analogs, GnRH I and II peptides and analogs, octreotide, depreotide, vapreotide, vasoactive intestinal peptide (VIP), cholecystokinin (CCK), RGD-containing

peptides, melanocyte-stimulating hormone (MSH) peptide, neurotensin, calcitonin, peptides from complementarity determining regions of an antitumor antibody, glutathione, YIGSR (leukocyte-avid peptides, e.g., P483H, which contains the heparin-binding region of platelet factor-4 (PF-4) and a lysine-rich sequence), atrial natriuretic peptide (ANP), β-amyloid peptides, delta-opioid antagonists (such as

ITIPP(psi)), annexin-V, endothelin, leukotriene B4 (LTB4), chemotactic peptides (e.g., N-formyl-methionyl-leucyl-phenylalanine-lysine (fMLFK)), GP Iib/IIIa

receptor antagonists (e.g., DMP444), human neutrophil elastase inhibitor (EPI-FINE-2 and EPI-HNE-4), plasmin inhibitor, antimicrobial peptides, apticide (P280 and P274), thrombospondin receptor (including analogs such as TP- 1300), bitistatin, pituitary adenylyl cyclase type I receptor (PAC1), fibrin a-chain, peptides derived from phage display libraries, and conservative substitutions thereof.

Immunoreactive ligands for use as the targeting moiety Z in the invention include an antigen-recognizing immunoglobulin (also referred to as "antibody"), or antigen-recognizing fragment thereof. As used herein, "immunoglobulin" refers to any recognized class or subclass of immunoglobulins such as IgG, IgA, IgM, IgD, or IgE. Preferred are those immunoglobulins which fall within the IgG class of immunoglobulins. The immunoglobulin can be derived from any species. Preferably, however, the immunoglobulin is of human, murine, or rabbit origin. In addition, the immunoglobulin may be polyclonal or monoclonal, but is preferably monoclonal.

Conjugates of the invention may include an antigen-recognizing

immunoglobulin fragment. Such immunoglobulin fragments may include, for example, the Fab', F(ab')₂, F_v or Fab fragments, or other antigen-recognizing immunoglobulin fragments. Such immunoglobulin fragments can be prepared, for example, by proteolytic enzyme digestion, for example, by pepsin or papain digestion, reductive alkylation, or recombinant techniques. The materials and methods for preparing such immunoglobulin fragments are well-known to those skilled in the art. See Parham, J. Immunology, 131, 2895, 1983; Lamoyi et al., J. Immunological Methods, 56, 235, 1983.

The immunoglobulin used in conjugates of the invention may be a "chimeric antibody" as that term is recognized in the art. Also, the immunoglobulin may be a "bifunctional" or "hybrid" antibody, that is, an antibody which may have one arm having a specificity for one antigenic site, such as a tumor associated antigen while the other arm recognizes a different target, for example, a hapten which is, or to which is bound, an agent lethal to the antigen-bearing tumor cell. Alternatively, the bifunctional antibody may be one in which each arm has specificity for a different epitope of a tumor associated antigen of the cell to be therapeutically or biologically modified. Hybrid antibodies thus have a dual specificity, preferably with one or more binding sites specific for the hapten of choice or one or more binding sites specific for a target antigen, for example, an antigen associated with a tumor, an infectious organism, or other disease state. Biological bifunctional antibodies can also be used as the Z targeting moiety in the conjugate compounds of the invention (such antibodies are described in, for example, European Patent Publication, EPA 0 105 360, which is hereby incorporated by reference in its entirety). Such hybrid or bifunctional antibodies may be derived either biologically, by cell fusion techniques, or chemically, such as with cross- linking agents or disulfide bridge-forming reagents, and may be comprised of whole antibodies and/or fragments thereof. Methods for obtaining such hybrid antibodies are disclosed, for example, in PCT application W083/03679, published Oct. 27, 1983, and published European Application EPA 0 217 577, published Apr. 8, 1987, each of which is hereby incorporated by reference in its entirety. Particularly preferred bifunctional antibodies are those biologically prepared from a "polydoma" or "quadroma" or which are synthetically prepared with cross-linking agents such as bis- (maleimido)-methyl ether ("BMME"), or with other cross-linking agents familiar to those skilled in the art.

In addition, the immunoglobin may be a single chain antibody ("SCA"). An

SCA may consist of single chain Fv fragments ("scFv") in which the variable light ("V_L") and variable heavy ("V_H") domains are linked by a peptide bridge or by disulfide bonds. Also, the immunoglobulin may consist of single VH domains (dAbs) that possess antigen-binding activity. See G. Winter and C. Milstein, Nature 349:295, 1991; R. Glockshuber et al., Biochemistry 29:1362, 1990; and, E. S. Ward et al., Nature 341 :544, 1989.

Especially preferred for use in the present invention are chimeric monoclonal antibodies; preferably those chimeric antibodies having specificity toward a tumor associated antigen. As used herein, the term "chimeric antibody" refers to a monoclonal antibody comprising a variable region, i.e. a binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies having a murine variable region and a human constant region are especially preferred in certain applications of the invention, particularly human therapy, because such antibodies are readily prepared and may be less immunogenic than purely murine monoclonal antibodies. Such murine/human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding murine immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions. Other forms of chimeric antibodies for use in conjugates of the invention are those in which the class or subclass has been modified or changed from that of the original antibody. Such "chimeric" antibodies are also referred to as "class-switched antibodies." Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. See Morrison, S. L, et al., Proc. Nat'l Acad. Sci., 81 :6851, 1984.

The term "chimeric antibody" also includes a "humanized antibody," namely, those antibodies in which the framework or "complementarity determining regions" ("CDR") have been modified to include the CDR of an immunoglobulin of different specificity, as compared to that of the parent immunoglobulin. In a preferred embodiment, a murine CDR is grafted into the framework region of a human antibody to prepare the "humanized antibody." See, e.g., L. Riechmann et al., Nature 332:323, 1988; M. S. Neuberger et al, Nature 314:268, 1985. Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for the chimeric and bifunctional antibodies. See, e.g., EPA 0 239 400 (published Sep. 30, 1987), which is hereby incorporated by reference in its entirety.

One skilled in the art will recognize that a bifunctional-chimeric antibody can be prepared which would have the benefits of lower immunogenicity of the chimeric or humanized antibody, as well as the flexibility, especially for therapeutic treatment, of the bifunctional antibodies described above. Such bifunctional-chimeric antibodies can be synthesized, for instance, by chemical synthesis using cross-linking agents and/or recombinant methods of the type described above. The present invention should not be construed as limited in scope by any particular method of production of an antibody whether bifunctional, chimeric, bifunctional-chimeric, humanized, or an antigen-recognizing fragment or derivative thereof.

Conjugates of the invention may also include, as the Z targeting moiety, immunoglobulins (as defined above) or immunoglobulin fragments to which are fused active proteins, for example, an enzyme of the type disclosed in Neuberger, et al., PCT application W086/01533, published Mar. 13, 1986, which is hereby incorporated by reference in its entirety.

As used herein, "bifunctional," "fused," "chimeric" (including humanized), and "bifunctional-chimeric" (including humanized) antibody constructions also include, within their individual contexts, constructions including antigen recognizing fragments. As one skilled in the art will recognize, such fragments may be prepared by traditional enzymatic cleavage of intact bifunctional, chimeric, humanized, or chimeric-bifunctional antibodies. In the event that intact antibodies are not susceptible to such cleavage, e.g., because of the nature of the construction involved, the noted constructions can be prepared with immunoglobulin fragments used as the starting materials; or, if recombinant techniques are used, the DNA sequences, themselves, can be tailored to encode the desired "fragment" which, when expressed, can be combined in vivo or in vitro, by chemical or biological means, to prepare the final desired intact immunoglobulin "fragment." It is in this context that the term "fragment" is used herein.

The immunoglobulin (antibody), or fragment thereof, used as the Z targeting moiety in conjugate compounds of the present invention may be polyclonal or monoclonal in nature. Monoclonal antibodies are the preferred immunoglobulins. The preparation of polyclonal or monoclonal antibodies is well known to those skilled in the art. See, e.g., G. Kohler and C. Milstein, Nature 256:495, 1975. In addition, hybridomas and/or monoclonal antibodies which are produced by such hybridomas and which are useful in the practice of the present invention are publicly available. Linkers for Attaching the Cytotoxic, Therapeutic, or Diagnostic Agents

("Cargo" or X moiety) to the Y and Z Moieties to Produce Conjugate

Compounds of the Invention

As discussed above, conjugate compounds of the invention can include cytotoxic, therapeutic, or diagnostic agents as "cargo" (e.g., nucleic acids molecules, PNAs, peptides, polypeptides, proteins, small molecules, antibodies, or antibody fragments). These agents can be associated with or bonded to the Y and Z moieties of the conjugate compound, which faciliate the delivery and targeting of the conjugate compound, respectfully, using, e.g., a linker or linking component.

In one embodiment, conjugate compounds of the invention are prepared by incorporating a peptidic linking group into the the X moiety (e.g., nucleic acid molecules, such as siRNA and dsRNA), PNA molecules, peptides, polypeptides, proteins, small molecules, antibodies, or antibody fragments). The peptide linking group can include, e.g., a cysteine residue that is used to form a disulfide bond between the X moiety and the Y moiety or it can be another peptide sequence that forms a bond that is cleavable by a known cytosolic enzyme.

Other types of linkers are also envisioned. For example, the linker can also couple the X moiety (i.e., the "cargo") to the Y moiety by reacting, e.g., a free amino group of a Thr residue of a peptide portion of X moiety to the conjugate compound (e.g., a portion of the Y moiety) with an appropriate functional group of a chelator, such as a carboxyl group or activated ester. For example, a conjugate may

incorporate the chelator ethylenediaminetetraacetic acid (EDTA), common in the art of coordination chemistry, when functionalized with a carboxyl substituent on the ethylene chain. Synthesis of EDTA derivatives of this type are reported in Arya et ah, Bioconjugate Chemistry, 2:323, 1991), wherein the four coordinating carboxyl groups are each blocked with a t-butyl group while the carboxyl substituent on the ethylene chain is free to react with the amino group of a peptide portion of the agent of the invention, thereby forming a conjugate.

Formulations of the Conjugate Compounds of the Invention

Conjugate compounds of the invention may be administed to a mammalian subject, such as a human, directly or in combination with any pharmaceutically acceptable carrier or salt known in the art for use in the treatment or detection of disease. Pharmaceutically acceptable salts may include non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences. (18^th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA.

Pharmaceutical formulations of a therapeutically effective amount of a conjugate compound of the invention, or pharmaceutically acceptable salt-thereof, can be administered orally, parenterally (e.g., by intramuscular, intraperitoneal, intravenous, or subcutaneous injection, by inhalation, intradermally, using optical drops, or by implant), nasally, vaginally, rectally, sublingually, or topically, in admixture with a pharmaceutically acceptable carrier adapted for the route of administration.

Methods well known in the art for making formulations are found, for example, in Remington's Pharmaceutical Sciences (18^th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA. Compositions intended for oral use may be prepared in solid or liquid forms according to any method known to the art for the manufacture of pharmaceutical compositions. The compositions may optionally contain sweetening, flavoring, coloring, perfuming, and/or preserving agents in order to provide a more palatable preparation.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier or excipient. These may include, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, sucrose, starch, calcium phosphate, sodium phosphate, or kaolin. Binding agents, buffering agents, and/or lubricating agents (e.g., magnesium stearate) may also be used. Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and soft gelatin capsules. These forms contain inert diluents commonly used in the art, such as water or an oil medium. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying agents, and suspending agents.

Formulations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, or emulsions. Examples of suitable vehicles include propylene glycol, polyethylene glycol, vegetable oils, gelatin, hydrogenated naphalenes, and injectable organic esters, such as ethyl oleate. Such formulations may also contain adjuvants, such as preserving, wetting, emulsifying, and dispersing agents. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylcne copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for the polypeptides of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

Liquid formulations can be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, or by irradiating or heating the compositions. Alternatively, they can also be manufactured in the form of sterile, solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately before use.

Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to active substances, excipients such as coca butter or a suppository wax. Compositions for nasal or sublingual administration are also prepared with standard excipients known in the art. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops or spray, or as a gel.

The amount of active ingredient in the compositions of the invention can be varied. One skilled in the art will appreciate that the exact individual dosages may be adjusted somewhat depending upon a variety of factors, including the type of conjugate compound being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the nature of the subject's conditions, and the age, weight, health, and gender of the patient. In addition, the severity of the condition targeted by the conjugate compounds of the invention may also have an impact on the dosage level. Generally, dosage levels of between 0.1 μg/kg to 100 mg/kg of body weight are administered daily as a single dose or divided into multiple doses. Preferably, the general dosage range is between 250 μg/kg to 5.0 mg/kg of body weight per day. Wide variations in the needed dosage are to be expected in view of the differing efficiencies of the various routes of administration. For instance, oral administration generally would be expected to require higher dosage levels than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, which are well known in the art. In general, the precise therapeutically effective dosage will be determined by the attending physician in consideration of the above identified factors. Administration of compositions of the invention may be as frequent as necessary to obtain the desired therapeutic effect. Some patients may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate. Other patients, however, receive long-term treatments at the rate of 1 to 4 doses per day, in accordance with the physiological requirements of each patient. The active product may be administered, e.g., intravenously, 1 to 4 times daily or via continuous infusion.

The conjuage compounds of the invention can be administered in a sustained release composition, such as those described in, for example, U.S.P.N. 5,672,659 and U.S.P.N. 5,595,760. The use of immediate or sustained release compositions depends on the type of condition being treated. If the condition consists of an acute or over- acute disorder, a treatment with an immediate release form will be preferred over a prolonged release composition. Alternatively, for preventative or long-term

treatments, a sustained released composition will generally be preferred.

Conjugate compounds of the present invention can be prepared in any suitable manner. The nucleic acid or polypeptide portions of the conjugate compounds may be isolated from naturally occurring sources, recombinantly produced, produced synthetically, or produced by any combination of these methods. The synthesis of short peptides and nucleic acids molecules is well known in the art. See e.g., Stewart et al., Solid Phase Peptide Synthesis (Pierce Chemical Co., 2d ed., 1984). The conjugate compounds of the present invention can be synthesized according to standard peptide synthesis methods known in the art.

Use of Toxins as a Platform to Deliver Cytotoxic, Therapeutic, Diagnostic Agents as Cargo

Toxins (e.g., the transmembrane and receptor binding domains of the DT) can be used as a platform to facilitate the highly efficient delivery of cargo (e.g., nucleic acid molecules, such as siRNA, polypeptides, or chemical compounds) to the cytosol of target eukaryotic cell s. The "cargo" affixed to the toxins can be easily substituted to yield multiple different delivery constructs. For example, the toxins can be engineered to include a cysteine residue at its amino-terminal end or an existing cysteine residue within the toxin (e.g., the cysteine residue located at amino acid position 201 of DT (see, e.g., Figure 1 A) can be used to "load" the cargo onto the toxin construct via the formation of disulfide bonds between the cargo (referred to herein as the "X" moiety) and the toxin platform (referred to herein as the

combination of the "Y" and "Z" moieties). Other types of covalent or ionic linkages known in the art can be employed to link the desired "cargo" to the delivery constructs, if desired.

In addition, the toxins can be prepared as fusion proteins so that they include a receptor binding domain substitution to include a receptor or ligand binding domain (referred to herein as the "Z" moiety) that will target the construct to a cell or population of cells expressing the receptor or ligand to which the Z moiety binding domain interacts. This will provide targeting specificity to the conjugate constructs of the invention. The cargo of the conjugate compounds may be, e.g., nucleic acid molecules (e.g., siRNA molecules), amino acids (e.g., polypeptides), or small molecules (e.g., cytotoxic agents) that can be delivered to a cell to treat a particular disease or disorder. The constructs of the inventio are designed to solve the problem of efficient delivery into the eukaryotic cell cytosol from the lumen of endosomal vesicles.

The diphtheria toxin structural platform as a nano-machine in the

development of a eukaryotic cell-specific membrane translocation delivery system.

In general, the evolution of bacterial protein toxins can be viewed as the natural assembly of highly efficient nano-machines capable of cell surface receptor specific delivery of their respective catalytic domains, "cargo", to the cytosol. The DT structural platform can be exploited to develop a system for the facilitated delivery of, e.g., nucleic acid molecules (e.g., siRNA), polypeptide therapeutics, or small molecules across the endosomal vesicle membrane and their subsequent release into the cytosol of target cells and tissues. The conjugate compounds of the invention deliver their "cargo" using the same mechanism by which native DT delivers its C- domain cargo to the eukaryotic cell cytosol.

The intoxication of sensitive eukaryotic cells by DT follows an ordered series of events. As shown in Figure 7, the first step in the intoxication process is the binding of toxin to its cell surface receptor, the heparin-binding (hb)-EGF-like precursor (Naglich el ah, 1992). This binding is further enhanced by the DT receptor associated protein DTRAP27, which is the primate homologue of human CD9 (Iwamoto et al., 1994). Receptor bound toxin is concentrated in clathrin coated pits and internalized into clathrin coated vesicles (CCVs), which are then converted into early endosomal vesicles (EEVs) (Moya et al., 1985). As the clathrin triskelon is replaced with a new set of protein components, including Arfl and COPI coatomer complex, the activity of the vacuolar ATPase lowers the luminal pH within the EEV. Acidification of the vesicle lumen is required to trigger the unfolding of the DT transmembrane domain (Boquet et al, 1976). As the luminal pH falls to between 5.5 - 5.0, the transmembrane domain undergoes a dynamic reorganization which results in the spontaneous insertion of two a-helical hairpins into the vesicle membrane resulting in the formation a transmembrane 18-25 A pore (Donovan et ah, 1981; Kagan et al., 1981). Pore formation is a critical step in the intoxication process since it provides the conduit for C-domain translocation from the cis to trans side of the endosomal vesicle membrane with subsequent events effecting C-domain release into the target cell cytosol.

Translocation of the DT C-domain from the endosomal vesicle lumen to the eukaryotic cell cytosol is facilitated by a Cytosolic Translocation Factor (CTF) complex. It is clear that the CTF complex is composed of at least COPI complex proteins, Hsp90, and thioredoxin reductase (Ratts et al., 2003; Ratts et al., 2005; Tamayo et al., 2008; Trujillo et al., submitted). Once the C-domain has been translocated through the pore formed by the transmembrane domain, the disulfide bond between the catalytic and transmembrane domains is reduced by thioredoxin reductase (Ratts et al., 2003) and the C-domain is released into the cytoplasm. It is well known that the DT C-domain is an ADP-ribosyltransferase that catalyzes the NAD+ -dependent ADP-ribosylation of eukaryotic elongation factor 2 (EF-2) thereby inhibiting cellular protein synthesis (Honjo et ah, 1968; Collier and Kandel, 1971 ; Gill and Pappenheimer, 1971). Upon cessation of protein synthesis the intoxicated cell will ultimately die by apoptosis (Kochi and Collier, 1993).

Decoding the rules that allow for efficient delivery of "cargo "

from the endosomal vesicle lumen to the eukaryotic cell cytosol.

Four critical findings have provided key insights into the rules that govern the delivery of, e.g., the DT C-domain and anthrax toxin LF into the cytosol of target cells. The first was made by Lemichez et al. (1997) who found that anti-P-COP antibodies blocked the in vitro translocation of the DT C-domain from the endosomal vesicle lumen to the external medium. This report was confirmed and extended by Ratts et al (2005) who demonstrated that at least β-COP was a component of the CTF complex. It has now been demonstrated that rather than β-COP alone, it is the entire COPI complex that is essential for C-domain delivery. And finally, the cytosolic delivery of anthrax LF also requires COPI complex proteins (Tamayo et al., 2008, and Abrami et al, 2004).

COPI complex is composed of seven proteins (α, β, β', γ, δ, ε, and ζ). While COPI complex has been well studied in its roll in vesicle recycling mechanisms associated with the movement of newly synthesized proteins through the secretory pathway, this complex has also been implicated in a variety of additional trafficking functions and has been demonstrated on endosomes. The requirement for COPI complex in both the delivery of the DT C-domain and anthrax LF to the eukaryotic cell cytosol has been shown. Ratts et al. (2005) identified the Tl motif in

transmembrane helix 1 of DT and demonstrated that this motif was essential in the C- domain entry process. It is of interest to note that the Tl motif is highly conserved in anthrax LF, anthrax EF, and the botulinum neurotoxins. I have also demonstrated that the Tl motif and its closely associated XKXX sequences mediate the facilitated delivery of anthrax LF into the eukaryotic cell cytosol (Tamayo et al., 2008). The Tl motif and its closely positioned KXKXX sequences closely resemble the dilysine (KKXX) and interrupted dilysine motifs (KXKXX) in the cytoplasmic tails of both p23/24 adaptor and cargo proteins (Cosson & Letourneue, 1994) that bind COPI coatomer complex proteins and direct vesicle trafficking (Fiedler et al., 1966). As shown in Figure 8A & B, multiple KXKXX sequences in the N-terminal portion of both the DT transmembrane domain and anthrax LF raises the possibility that sequential association/disassociation events between Arfl-GTP::KXKXX::COPI in, e.g., the DT C-domain and anthrax LF result in a "ratchet-like" entry process. In the case of the DT C-domain the reduction of the disulfide bond between the

transmembrane and C-domains by thioredoxin reductase and refolding of the C- domain by Hsp90 (Ratts et al., 2003) and Hsc70 results in the release of the cargo (i.e., in native DT, the enzymatically active fragment A) into the target cell cytosol.

The following rules apply for diphtheria toxin-mediated delivery of its native catalytic domain "cargo" from the lumen of endosomal vesicles to the eukaryotic cell cytosol, and thus also apply to the delivery of other "cargo" (i.e., the X moiety of the conjugate compounds of the invention) that is substituted for the native fragment A of DT:

(1) Specific binding of the toxin and its "cargo " to a cell surface receptor that is internalized into an endosomal compartment that becomes acidified;

(2) Low pH triggered insertion of the transmembrane domain into the

endosomal vesicle membrane to form a pore through which the "cargo " is threaded by the chaperone-like properties of the transmembrane domain: and, upon the emergence of transmembrane helix 1 sequences on the cytosolic surface of the endosomal vesicle membrane,

(3) The transmembrane domain Tl motif and/or KXKXX sequences (e.g., dibasic signature (e.g., KKXX, KXKXX) and/or an aromatic amino acid sequences (e.g. FFXXBB(X)„) that function as p23/24 adaptor mimetics and mediate the binding of COPI complex proteins then facilitate the delivery of the catalytic domain "cargo " into the cytosol; the cargo, which is bound to the transmembrane domain and receptor binding domain via a disulfide bond is then reduced by thioredoxin reductase, thereby releasing the cargo into the cytosol.

Diphtheria Toxin and Diphtheria Toxin-Based Fusion Protein Toxins

DT is synthesized as a precursor and following cleavage of its 25 amino acid signal sequence, is released into the culture medium as a 535 amino acid protein (Smith et al, 1980; Greenfield et al., 1983; Kaczorek et al., 1983). The ADP- . ribosyhransferase activity of the toxin is activated by the endoprotease furin "nicking" of the a-carbon backbone at Argl93 in an exposed 14 amino acid loop formed by a disulfide bond between Cysl86 and Cys201. Upon reduction under denaturing conditions, "nicked" toxin may be separated into a 21.1 kDa N-terminal polypeptide (residues 1-193), or Fragment A, and a 41.2 kDa C-terminal Fragment B (residues 194 to 535), which carries both the transmembrane and receptor binding domains (Uchida et al, 1971; Choe et al, 1992; Bennett et al, 1994). Once delivered to the eukaryotic cell cytosol, the C-domain catalyzes the NAD⁺-dependent ADP- ribosylation of elongation factor 2 (EF-2), which results in the inhibition of cellular protein synthesis and ultimately cell death by apoptosis (Collier and Kandel, 1971 ; Gill and Pappenheimer, 1971 ; Kochi and Collier, 1993).

In 1986, DT was shown to be amenable to use as a structural platform to genetically construct a family of fusion proteins toxins (Murphy et al, 1986;

vanderSpek and Murphy, 2000). Using protein engineering methods these chimeric toxins were constructed at the level of the gene by deletion of those sequences encoding the native receptor binding domain and their replacement with

oligonucleotide sequences which encoded a surrogate receptor binding domain. Each of these chimeric fusion protein toxins were found to be selectively toxic toward only those cells which carried the targeted cell surface receptor. For example, the fusion protein toxin DAB₃g9lL-2 (ONTAK^®), which targets the high affinity form of the IL-2 receptor is highly potent (IC50 = 1 - 5 pM) against high affinity IL-2 receptor bearing cells; whereas, it is essentially non-toxic (IC50 = 1 - 10 μΜ) for cells that do not express this receptor. It should be noted that DAB₃₈₉lL-2 was the first, and to date only, targeted toxin to be approved by the FDA for human clinical use (Foss, 2000). As shown in Table 2, a series of selectively toxic fusion proteins have been constructed by substitution of the native diphtheria toxin receptor binding domain with a variety of different growth factors and cytokines. In each instance these individual fusion protein toxins are selectively active against only those eukaryotic cells that express the targeted cell surface receptor.

Table 2

Fusion Protein Toxin Targeted Receptor Cytoxicity (IC^

DAB₃₈₉IL-2 IL-2R 3 x 10^"11 M

DAB₃₈₉aMSH a-MSH 1 x 10-¹²

DAB₃₈₉IL-3 IL-3R 5 x 10-¹² M

DAB₃₈₉IL-4 IL-4R 2 x 10- ⁰ M

DAB₃₈₉IL-6 IL-6R 2 x 10-¹¹ M

DAB₃₈₉IL-7 IL-7R 1 x 10-¹⁰ M

DAB₃₈₉GMCSF GMCSFR 3 x 10^"11 M

DAB₃₈₉EGF EGFR 1 x 10^"12

DAB₃₈₉CD4 HIV gp120 1 X 10-9 M

As is evident from the above, the DT structural platform is remarkably amenable to receptor binding domain substitution, thereby allowing this domain of DT to be substituted with a range of desired binding domain (e.g., a receptor, the receptor ligand, and other binding molecules, such as antibodies or antibody fragments, that can be used to target the conjugate compound to a desired cell or tissue). This detailed understanding of the structural features of the DT

transmembrane domain that are required for COPI complex facilitated delivery of "cargo" from the lumen of acidified endosomal vesicles to the cytosol allows for the use of the DT structural platform for the development of a system to efficiently deliver cytotoxic, therapeutic, or diagnostic agents (e.g., nucleic acid molecules, such as siRNA and PNA molecules, polypeptides, such as growth and transcription factors, cytotoxic agents, such as paclitaxel, and anti-viral agents) to the cytosol of target cells. This system envisions the use of the DT B-fragment transmembrane and receptor binding domains (and substitutions thereof) for (i) the targeting of specific cell surface receptors or ligands for cell-specific and/or tissue specific delivery, and (ii) the transmembrane domain for endosomal vesicle membrane pore formation and COPI complex binding for the facilitated delivery of siRNA "cargo" to the target cell cytosol. Delivery of siRNA molecules using Conjugate Compounds of the Invention

RNAi is well known to be a fundamental pathway in eukaryotic cells. This pathway is triggered by the presence of long pieces of double stranded RNA (dsRNA) (Elbashir et al., 2001). The cytoplasmic nuclease Dicer is known to cleave these long segments of dsRNA into 21-23 nt pieces of double stranded RNA (siRNA) (Bernstein et al., 2001). siRNA is then incorporated into the RNA-induced silencing complex (RISC) where the dsRNA becomes unwound (Rand et al., 2004; Matranga et al., 2005). Upon degradation of the sense strand, the antisense RISC complex then seeks out and degrades the messenger (m)RNA that is complementary to the antisense strand (Ameres et al., 2007). Since the antisense siRNA-RISC complex is stable, mRNA that is targeted continues to be degraded over time. In fact, siRNA mediated knockdown of mRNA is known to last for 3-7 days in rapidly dividing cells, and for weeks in non-dividing cells (Bartlett & Davis, 2006).

The mode of action of siRNA and the fact that a specific siRNA can be designed to knockdown expression of virtually every gene in an organism offers enormous potential for the development of gene specific therapy for human disease. In fact, specific siRNAs have been developed for hepatitis B virus (Morrissey et al., 2005); human papilloma virus (Niu et al., 2006), liver cirrhosis (Sato et al., 2008), ovarian cancer (Haider et al., 2006), and bone cancer (Takeshita et al., 2005); the siRNA molecules described in the publications and others can be incorporated into the conjugate compounds of the invention to treat these and other diseases.

While the promise is great, significant technical barriers exist for the development of siRNA as a clinical therapeutic. As is discussed below, these barriers are overcome by the conjugate compounds of the invention. For example, native oligonucleotide constructs are known to be unstable in cellular environments because of their susceptibility to degradation by endogenous nucleases (Eckstein, 2007). Some progress has been made in mitigating this problem through the use of phosphothionate and morpholino nucleic acid analogs in the synthesis of siRNA constructs (Sazani et al., 2001). The replacement of native phosphate with phosphothionate linkages has resulted in siRNAs with increased nuclease resistance, but this approach has also resulted in increased non-specific binding to cellular proteins. Morpholino linkages which are non-ionic have proven to be more effective since they do not interact with cellular proteins, and as a result have proven to result in siRNAs that are more stable and specific. Despite these advances, the major technical hurtle, the cytoplasmic delivery of siRNAs, was not addressed until the development of the conjugate compounds of the invention.

The cytoplasmic delivery of siRNAs in vitro have been shown to require one of a variety of transfection methods: linkage to cell penetrating peptides (Turner et al., 2007; Deshayes et al., 2008; Lebleu et al., 2008); conjugation to cholesterol

(Soutschek et al., 2004; Cheng et al., 2006); association with proteamine-antibody fusion proteins (Song et al., 2005; Vornlocher, 2006), use of atelocollagen

(Minakuchi et al., 2004; Takashita et al., 2005), construction of stable nucleic acid- lipid particles (Monissey et al., 2005; Santel et al., 2006); polyethyleneimine (Ge et al.,2004; Grzelinski et al., 2006); and encapsulation in liposomes (Feigner et al., 1987; Malone et al., 1989). While many of these methods are effective in vitro, their use in vivo remains problematic.

Since RNAi is a cytoplasmic process, efficient delivery to the cytosol following either adsorption to the cell surface or from the lumen of an endosomal vesicle after fluid phase endocytosis is absolutely essential. Some progress has been made with phosphorothionate-mediated uptake of naked siRNA via the caveosomal uptake pathway (Overhoff & Sczakiel, 2003). However, even by this route of entry large amounts of siRNA are internalized, but the bulk of the internalized siRNA remains in perinuclear vesicles, and as a result the knockdown of target gene expression is limited (Mescalchin et al. 2007).

Cellular delivery of siRNA using conjugate compounds of the invention:

Conjugation and analysis of peptide-PNA and fragment B197 conjugates.

A diphtheria toxin-based system can be used to facilitate cytosolic delivery of oligonucleotide antisense cargo. For example, peptide-PNA can be conjugated with Fragment B from the non-toxic mutant of diphtheria toxin, CRM 197. The approach is based upon methods that were developed for the reconstitution of "native" diphtheria toxin from two non-toxic mutants, CRM 197 and CRM45 (Uchida et al., 1973).

(a) Conjugation of purified Fragment B197 with peptide- PNA: The conditions for reassembly in high yield for the "reconstitution" of fully active diphtheria toxin from two non-toxic mutants, CRM45 and CRM 197, have been described. CRM45 contains a fully active A fragment; whereas, CRM 197 carries a defective A fragment and a fully functional B fragment of the toxin (Uchida et al., 1973).

Fragment B197 can be used in the assembly of disulfide bond linked conjugate of peptide-PN A/fragment B 197 as shown in Figure 11. The PNA portion of the conjugate encodes an antisense oligomer that is designed to block the aberrant IVS2- 654 splice site in intron-2 of the luc gene (see Fig. 9). The peptide portion of the conjugate encodes 6 amino acids upstream of Cysl86 as well as the downstream sequence which terminates at the end of the furin recognition/cleavage site RVRR- COOH. In this construct, the reactive sulfhydryl moiety of Cysl86 is used to form a disulfide bond to fragment B197.

Fragment B197 can be purified from CRM197 according to the method of Uchida et al. (1973). Briefly, purified CRM197 (List Biological Laboratories, Campbell, CA) is treated with immobilized trypsin for 10 min at 37°C in the presence of 10 mM dithiothreitol in 0.02M Tris-HCl buffer at pH 8.0. The reaction is stopped by centrifuging the reaction mix through a spin column. This method allows the separation of the cleaved ppolypeptides from the immobilized trypsin which does not pass through the column frit. Under denaturing conditions the trypsin treated CRM 197 is then be purified by HPLC sizing in order to separate fragment A197 from B197. Purified fragment B 197 is then be mixed with an excess concentration of reduced peptide-PNA. The molar ratio of peptide-PNA to fragment B197 for can be varied (e.g., 1 : 1, 5:1; 10: 1 ; 20:1, 100:1 ; 500:1), as necessary. The mixture is then be dialyzed against 0.01M phosphate buffer, pH 7.2, for 24 hrs at 4°C to remove dithiothreitol and permit disulfide bond formation. The disulfide linked peptide- PN A/B 197 conjugate is then be purified from free peptide-PNA by HPLC sizing and/or dialysis.

(b) Peptide-PNA-Fragment B197 mediated correction of Luc-IVS2-654 aberrant splicing in CHO-K1 cells: The delivery of peptide-PNA to the cytosol of Luc-/ 5'2-654 CHO-K1 cells can be performed essentially as previsouly described (Wright, Zhang, & Murphy, Biochem. Biophys. Res. Commun. 376:200-205, 2008). Briefly, frozen cell stocks are thawed and cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum in 12- and 24-well plates for up to 72 hrs. Since these cell lines grow in monolayers, the seeding concentration (~ 1-5 x 10⁴/ml) can be adjusted so that the monolayer does not become confluent during the experimental culture period. Cells can be treated with varying concentrations (10^~8- 10^"I2M) of peptide-PNA-Fragment B197 conjugate for 1 hr and then washed to remove unbound material. Following 24 and 36 hr incubation at 37°C in a 5% C0₂ atmosphere, cells will be harvested, lysed, and total cell protein can be assayed for luciferase activity. In addition, cells can be harvested, lysed, and total RNA will be extracted Trizol (Invitrogen) for RT-PCR analysis of β-globin mRNA. Since CHO- Kl cells express the hb-EGF-like precursor receptor for diphtheria toxin, the peptide- PNA-Fragment B197 conjugate should readily bind to the cell surface and be internalized into an early endosomal compartment.

In order to demonstrate that the cytosolic delivery of peptide-PNA and subsequent expression of luciferase was mediated by Fragment B197, the following controls can be used: (i) peptide-PNA alone should not, by itself, be delivered and therefore should not give rise to luciferase expression; (ii) CRM 197 can be used as a competitive inhibitor of peptide-PNA-Fragment B197 binding to the hb-EGF-like precursor receptor; (Hi) Bafilomycin Al can be used as an inhibitor of the vesicular ATPase in order to establish that vesicle acidification (required for Fragment B transmembrane domain insertion and pore formation in the endosomal membrane) is required for delivery of the peptide-PNA into the target cell cytosol. Finally, (iv) Trizol extracted RNA can be used for RT-PCR to confirm the expression of full length luc mRNA. In the latter experiment, RNA extracted from Luc-IVS2 CHO-Kl cells can be used as the positive control for RT-PCR. In these experiments the following primers which hybridize to sequences flanking Luc-IVS2 introns can be used: TTGATATGTGGATTTCGAGTCGTC and

TGTCAATCAGAGTGCTTTGGCG.

Previous results show that anthrax protective antigen markedly enhanced the delivery of PNA-Lyss to the eukaryotic cell cytosol (Wright, Zhang, & Murphy, 2008). The peptide PNA-Fragment B197 conjugate compound should also facilitate delivery of peptide-PNA to the cytosol of Luc-IVS-654 CHO-Kl cells. Upon delivery to the cytosol, the peptide-PNA should promote expression of full length luciferase; this can be confirmed by using, e.g., Oligofectamine (Invitrogen) to stimulate the cytosolic delivery of peptide-PNA as a positive control.

Diphtheria fragment B be used to successfully deliver a "chemically unique" PNA cargo.

The DT platform described herein can be used to deliver to the cytosol of a target cell a chemically unique PNA cargo. It is known that N-terminal extension fusion proteins of fragment A which include a duplicate A fragment and some of apolipoprotein Al can be delivered into the eukaryotic cell cytosol (Madhus et al., 1992). Thus, other cargo, such as a peptide-PNA (see Fig.l 1) disulfide cross-linked to fragment B of diphtheria toxin, can be delivered into the eukaryotic cell cytosol.

An alternative construct may be to cross link a PNA-Cys to a modified Fragment A (A197). In this instance, a Cys residue can be inserted at the N-terminal end of fragment A 197 in order to form a construct that is analogous to those described by Madhus et al. (1992). The recombinant protein can be expressed, purified and used to make the conjugate PNA-Cys-S-S-CRM197.

Conjugation and analysis of peptide-siRNA and fragment B197.

(a) Conjugation of purified Fragment B197 with peptide-siRNAs: The conjugation of peptide-siRNA to Fragment B197 can be accomplished using the same protocol as discussed above for the formation of peptide-PNA conjugate compounds, as described above. Briefly, peptide-siRNAs can be synthesized as 21 nt overlapping sense and antisense strands (BioSynthesis Inc., Lewisville, TX). In the first instance, the sense or antisense strand can be modified with a 3'-thiopropyl moiety that can be used for peptide coupling (this same modification can be used to couple any of the nucleic acid molecules described herein as "cargo" to the Y moiety for formation of the conjugate compounds of the invention). Under these conditions, after unwinding the antisense strand of the siRNA will be unmodified in the RISC-siRNA activated complex.

The peptide-PNAs and peptide-siRNAs can be synthesized chemically using techniques known in the art. (b) Peptide-siRN A/Fragment B 197 mediated silencing of Luc gene expression in CHO-Kl cells: As described above, protective antigen PA63 mediated the effective delivery of peptide-PNA and correction of aberrantly spliced

thalassemia-like IVS 654 β-globin luciferase reporter mRNA in Luc-IVS654 CHO-Kl cells. A peptide-siPvNA-fragment Bl 97 conjugate compound is expected to be equally successful. The control CHO-Kl Luc-IVS2 cell line, which expresses fully processed luciferase mRNA and exhibits luciferase reporter activity, can be used as a control. As shown in Figure 12, in both the Luc-IVS654 and Luc-IVS2 control cells, both the control and thalassemic-like mRNAs can be readily detected using RT-PCR. Firefly luciferase gene expression can be determined by quantitative RT- PCR using primers specific to the luciferase gene (forward 5'-

GCCTGAAGTCTCTGATTAAGT-3 ' , reverse 5'- ACACCTGCGTCGAAGT-3 ') and mRNA isolated from siRNA treated and control samples and correlated to direct measurements of luciferase activity using the Promega luciferase assay system with RLU determined by Wallac Microbeta, Victor2 (Perkin Elmer) counter or Turner Designs luminometer.

Bartlett and Davis (2006) used a luciferase reporter to characterize the kinetics of siRNA delivery into cells both in vitro and in vivo. Two sets of peptide- siRN As have been developed, as shown in Figure 13 for the construction of peptide- siRNA/fragment B 197 conjugates.

The A-form of RNA can be thread through the pore formed by the fragment B197 transmembrane domain

Wang et al. (2009) have recently described a method for the determination of helical RNA structure using NMR and small angle-X-ray scattering. Using these methods, Wang and colleagues report that A-form RNA in either parallel or anti- parallel duplexes has an envelope width of 22A. Barone et al. (2000) have measured the hydrodynamic radius of PNA, RNA, & duplex RNA, the latter of which has been measured to have a hydrodynamic radius of 12.8A. The pore formed by

transmembrane domain helices 5-9 of diphtheria toxin has been measured by Kagan et al. (1981) to be >18A and by Zalman & Wisnieski (1984) to be 24A in diameter. Based upon these reports the pore formed in the endosomal vesicle by the diphtheria toxin transmembrane domain is of sufficient size to allow the passage of peptide- siRNA and peptide PNA from the vesicle lumen to the cytosol.

The disulfide bond between the cargo (peptide-siRNA and peptide-PNA) and fragment B197 can be reduced in the endosomal vesicle lumen

It is well known that native diphtheria toxin may be "nicked" by proteases either during its purification from culture filtrates of Corynebacterium diphtherias, in serum, or on the cell surface by the endoproteinase furin. In fact, both native diphtheria toxin and the diphtheria toxin-related fusion protein toxins must be nicked in the protease sensitive loop between the A and B fragments into or to deliver the A fragment to the target cell cytosol. However, the A and B fragment of nicked diphtheria toxin are known to remain disulfide bond cross-linked until the fragments emerge from the transmembrane pore and are presented on the cytosolic surface of the endosomal vesicle and into the reducing environment of the cytosol. Ratts et al., (2003) demonstrated that the disulfide bond linking the A and B fragment of the toxin are reduced in the cytosol by thioredoxin reductase. Based upon these studies, the peptide-siRNA fragment B197 conjugate and the peptide-PNA fragment B197 conjugate are expected to remain disulfide bond cross-linked until the peptide-siRNA is delivered into the cytosol.

A bacterial protein toxin can facilitate the cytosolic delivery of antisense oligon ucleotide.

The receptor binding and transmembrane domains of anthrax protective antigen has been used as a vehicle for cell surface binding and endosomal vesicle pore formation. It is well known that anthrax protective antigen (PA83) binds to its cell surface receptors capillary morphogenesis gene 2 (CMG2) and tumor endothelial cell marker 8 (TEM8) (Bradley et al., 2001 ; Scobie et al., 2003). Once bound to its receptor, PA83 is cleaved to PA63 by the endoprotease furin (Klimpel et al., 1992; Gordon et al., 1995) and then reorganizes into a homo-heptamer (Milne et al., 1994). Each heptamer is then able to bind up to three molecules of Lethal Factor and/or Edema Factor. Anthrax toxin is then internalized in clathrin coated pits which become early endosomal vesicles. Upon acidification of the endosomal lumen, PA63 undergoes a dynamic change and spontaneously inserts into the vesicle membrane forming a pore. The mechanism of Lethal Factor entry into cytosol parallels that of diphtheria toxin and requires COPI complex binding to one or more of multiple KXKXX sequences and a Tl-like motif in the N-terminal region of the protein (Tamayo et al, 2008).

Since PA63 was known to specifically bind bind polylysine residues (Blanke et al., 1996), a polylysine (Lys₈) tail was attached to peptide nucleic acid through a 2'- o-methyl linkage (BioSynthesis Inc., Lewisville, TX). The sequence of the peptide nucleic acid was designed to hybridize to a modified luciferase gene which carries a human thalassemia β-globin gene intronic insert (Luc-IVS2-654) as shown in Figure 3. The assay system that was used to measure the delivery of peptide-PNA to the cytosol is shown in Figure 9, and was used previously to study the delivery of antisense analogs (Sazani et al., 2001). Using this system, the ability of anthrax protective antigen to facilitate the delivery of PNA-Lys₈ from the lumen of endosomal vesicles to the cytosol of CHO-K1 Luc-IVS-654 cells was examined. CHO-K1 cells were transfected with a plasmid that carries a luciferase gene containing a human thalassemia β-globin gene intronic insert {Luc-IVS-654). This intron encodes an alternative splice site that allows incorrect splicing of pre-mRNA transcripts, thereby preventing the translation of full length luciferase. However, the cytosolic delivery and binding of antisense oligonucleotides to the aberrant splice site in IVS-654 gives rise to correct splicing of β-globlin pre-mRNA and allows the translation of full length functional luciferase (Fig. 9). It should also be noted that anthrax protective antigen, in the absence of either anthrax lethal factor or edema factor, is not toxic toward eukaryotic cells.

It was expected that PA63 would facilitate both the delivery of PNA-Lys₈ through the PA63 pore and its release into the eukaryotic cell cytosol. In these experiments, CHO-K1 Luc-VIS-654 cells were exposed to PNA-Lys₈ in the absence or presence of either PA83 or PA63 and measured cytosolic delivery by induction of functional luciferase activity by measuring both chcmiluminescence and luc mRNA.

As can be seen in Figure 10, exposure of target cells to 0.3 μΜ antisense PNA-Lys₈ fails to give rise to luciferase activity that is above background; whereas, in the presence of either PA83 or PA63 exposure of target cells to 0.3 μΜ PNA-Lys₈ results in a marked enhancement of luciferase activity with a concomitant conversion of aberrantly spliced to correctly spliced luc mR A (Wright, Zhang, & Mu hy, 2008).

In earlier work with this system, Sazani et al. (2001) reported that PNA-Lys₄ alone was able to give rise to a modest stimulation of full length luciferase expression in μΜ concentrations. It was later confirmed that, at concentrations of 1.0 μg/ml PNA-Lys₈ alone gave rise to a slight increase in luciferase expression (Wright, Zhang, & Murphy, 2008). Whereas, in the presence of either protective antigen PA83 or PA63, the addition of 30 ng/ml PNA-Lys₈ gave rise to marked increase in the expression of full length luciferase (Fig. 10). Thus, protective antigen facilitated delivery of PNA-Lysg to the cytosol of target cells appears to be at least 50 -100- times, and perhaps as much as 1 ,000-times more efficient than PNA-Lys₈ alone.

These experiments confirm that delivery of peptide nucleic acid can be achieved by exploiting the cell surface receptor binding, endosomal membrane pore forming, and facilitated translocation of cargo from the endosomal vesicle lumen to the cytosol through the use of a bacterial protein toxin (in this case anthrax protective antigen).

Results

Based upon earlier observations describing the role that the Tl motif and transmembrane helix 1 arc likely to play in the catalytic domain entry process and the well defined cargo motifs recognized by COPI complex proteins, a site-directed mutational analysis of the multiple lysine residues in this region of diphtheria toxin was initiated. Figure 1 A shows a partial amino acid sequence of diphtheria toxin fragment B from Serine] 95 to Asparagine₂₃₅ showing transmembrane helices 1 and 2, the Tl motif and the multiple lysine residues that were anticipated to be most likely to mediate binding to COPI complex proteins and thereby facilitate catalytic domain entry. Since cloning of wild type DT is prohibited, site-specific mutations were introduced into the structural gene encoding the fusion protein toxin DAB₃₈9lL-2. DAB₃₈₉IL-2 is composed of native DT catalytic and transmembrane domain sequences, amino acids 1 -389, to which human interleukin 2 is genetically fused in the correct translational reading frame (Williams et ah, 1990). DAB₃₈₉lL-2 has been shown to be selectively toxic toward eukaryotic cells which display the high affinity IL-2 receptor, and except for receptor binding specificity the fusion protein toxin has been shown to follow the same route of entry into the cell as native diphtheria toxin (Bacha et a/., 1988).

A single K—> A mutations was first introduced at positions 213, 215, 217, and 222, after which the mutant fusion protein toxins were expressed in recombinant E. coli and partially purified as previously described (vanderSpek et al., 1993).

Individual mutant fusion proteins were then examined for cytotoxic potency by dose response analysis using high affinity IL-2 receptor bearing HuT102 cells as previously described (vanderSpek et al., 1993). As shown in Figure IB, the introduction of alanine site-directed substitution mutations at K213, K217, and K222 results in a loss of 1 - 2-logs of cytotoxic potency relative to the wild type fusion protein toxin. In marked contrast, the substitution of alanine for K215 results in a mutant which retains full cytotoxic activity. Lysine₂i₅ is positioned in the middle of the palindrome KTKTK, and the mutant DAB(K215A)₃₈9iL-2 still retains three lysine residues within transmembrane helix 1.

These observations suggested that within transmembrane domain helix 1 a combination of at least three lysine residues is most likely to be required for binding to COPI complex proteins and subsequent delivery of the C-domain. Furthermore, the spacing between these residues appears to affect the efficiency of C-domain entry into the cytosol as reflected by the 2-log range in cytotoxic potency of this group of single K→ A mutants. If this were the case, then either the introduction of any pair of K→ A mutations or the quadruple K→ A mutation in transmembrane helix 1 should lead to a complete loss of cytotoxic activity. In order to test this hypothesis, double mutants DAB(K213A, K215A)₃₈₉IL-2 and DAB(K215A, K217A)₃₈₉IL-2, and the quadruple mutant DAB(K213A, K215A, K217A, K222A)₃₈₉IL-2 were constructed. Following expression and purification, each mutant fusion protein was then assayed for cytotoxic activity. As anticipated, both of the double K→ A mutants as well as the quadruple K→ A mutant were found to be non-toxic (IC₅₀ ^> 5 x 10^"7M) (Figs.lC & ID).

Pull down experiments previously demonstrated that transmembrane helix 1 and the Tl motif mediate binding to at least β-COP of the COPI complex. Thus, additional tests were performed to determine whether it was the amino acid sequence of transmembrane helix 1 and the Tl motif or simply the ability to bind to COPI complex binding that was the essential feature to mediate the cytosolic delivery of the catalytic domain. Since the COPI binding motifs of Golgi and ER cargo and adaptor proteins have been well characterized, a domain swap mutant was constructed in which the 13 amino acid COPI binding segment from the cytoplasmic tail of the p23 adaptor protein was used to replace that portion of transmembrane helix 1 which contains the multiple lysine residues essential for catalytic domain delivery to the cytosol.

The structural gene encoding DAB₃₈₉IL2 was modified such that amino acids 212 - 223 which encompasses transmembrane helix 1 was deleted and replaced with the 13 amino acid sequence encoding the COPI binding portion of the p23 cytoplasmic tail (REILKKAKFFRRL). Following its genetic construction, the plasmid encoding the mutant toxin DAB(212p23) ₃8₉IL-2 was cloned, sequenced to verify correct insertion and reading frame of the COPI binding segment, and the recombinant mutant protein was expressed and purified as described in Experimental procedures below. As shown in Figure 2, dose response analysis of

DAB(212p23)₃₈₉IL-2 on Hutl02 cells is identical to that of the wild type DAB₃₈₉IL-2 (IC50 ^¾ 10 pM). The functional equivalence between the lysine-rich transmembrane helix 1 and the COPI binding segment from the cytoplasmic tail of the p23 adaptor protein suggests maintenance of the COPI complex binding function of this region is an essential feature in the catalytic domain entry process.

GST pull down experiments and precipitation assays were used to examine the ability of wild type and mutant transmembrane helix 1 sequences to interact with COPI complex proteins. It has been previously demonstrated both in vitro and in vivo that dilysine, poly-arginine and diphenylalanine motifs in the cytoplasmic regions of Golgi and ER cargo proteins play an essential role in recognition and binding by COPI complex pro teins. The proximity of numerous basic amino acids within transmembrane helix 1 of diphtheria toxin prompted an examination of the potential role played by these sequences in mediating the binding of COPI.

A series of pull down experiments were conducted in order to determine which subunit of the COPI complex was likely to facilitate the binding between transmembrane helix 1 sequences and the complex. The structural genes for γι-COP, β'-COP and ε-COP were expressed individually in vitro using a coupled transcription / translation rabbit reticulocyte lysate system. As shown in Figure 3, after in vitro synthesis and co-incubation with either GST or GST-DT140-271 , only GST-DT140- 271 specifically captured [ S]-labeled y COP. In contrast and in a manner consistent with other reports describing COPI binding using either cargo or adaptor proteins, the selective pull down of detectable amounts of either [³⁵S]-s-COP or [^J5S]- β'-COP using GST-DT 140-271 as bait was unsuccessful. These results suggest that both y!-COP in addition to the previously identified β-COP (Ratts et al, 2005), but neither β'- nor ε-COP are likely to mediate the protein-protein interaction(s) with transmembrane helix 1.

Since the results from both mutational analysis and GST-DT 140-271 pull down experiments suggested that residues in transmembrane helix 1 interacted with COPI in a fashion that was similar to the interactions between the complex and both cargo and p23 adaptor proteins, an analysis of the ability of individual peptides from this region to bind to COPI complex proteins was conducted (Table 3).

Table 3: Synthetic peptides used in this study.

Peptide Amino acid sequence MW

DTB5 TKTKIESLKEHGPIKNKM 2,081

KNOFF TATAIESLKEHGPIKNKM 1 ,967

KCOFF TKTKIESLKEHGPIANAM 1,967

KOFF TATAIESLAEHGPIANAM 1,796

wtp23 EILKKAKFFRRLHGPIKNKM 2,454

In order to conduct these experiments, an assay was used that was originally described by Hudson and Draper (1 97) and subsequently used by Reinhard et al. (1999) to demonstrate that paired diamines in neomycin, as well as the dibasic motifs contained within the cytoplasmic tails of p23/24 adaptor proteins [FFXXBB(X)_n] are capable of interacting with at least two specific sites on COPI complex, and depending upon the spacing induce aggregation and precipitation of COPI complex in vitro. Thus, to demonstrate that lysine residues in transmembrane helix 1 functioned like p23 adaptin and cargo motifs in their interaction with COPI, this precipitation assay was used to examine the ability of diphtheria toxin transmembrane domain helix 1 based peptides to bind to and precipitate COPI complex proteins. Figure 4A shows that DTB5, a synthetic peptide corresponding to amino acid residues 214 - 230 of the transmembrane domain, is able to induce aggregation and precipitation of COPI coatomer in vitro. In a similar fashion, the addition of increasing concentrations of the COPI binding peptide derived from the cytoplasmic tail of the p23 adaptor protein to the reaction mixture also resulted in the dose dependent precipitation of the complex in vitro (Fig. 4C).

In order to demonstrate that the ε-amino moieties of the lysine residues in DTB5 mediated the binding to COPI complex proteins, 1,3- cyclohexanebis(methylamine) (CBM) was added to the reaction mixture in increasing concentrations. As shown in Figure 4B, the addition of the diamine containing CBM to the reaction mixture completely blocked the DTB5 dependent precipitation of COPI complexes in vitro.

To further show that at least two pairs of lysine residues were necessary to induce COPI aggregation and precipitation, a series of DTB5 -related peptides were tested, in which pairs of lysine residues on either the N- or C-terminal ends were changed to alanine (KNOFF and KCOFF) or a peptide (KOFF) in which all 5 lysine residues were changed to alanine in the precipitation assay. As anticipated, removal of either all lysine residues or lysine residues from cither end of the peptide resulted in the complete loss of COPI complex aggregation and precipitation in vitro (Fig. 5).

Discussion

Lemichez et al. (1997) were the first to describe an in vitro assay to investigate the requirements for translocation of the diphtheria toxin catalytic domain from the lumen of acidified endosomal vesicles to the external medium. In this assay system, endosomal vesicles were pre-loaded with native diphtheria toxin in the presence of Bafilomycin Al, and the early endosomal vesicle enriched fraction was isolated by sucrose density gradient ultracentrifugation. Upon removal of

Bafilomycin Al, the addition ATP to the reaction mixture allowed acidification of the endosomal lumen; however, acidification of the lumen was not sufficient by itself to affect catalytic domain delivery to the external medium. The addition of both ATP and crude eukaryotic cytosolic extracts to the assay mixture was found to be essential for catalytic domain translocation across the vesicle membrane and its release into the external medium. In addition, Lemichez et al. (1997) found that the addition of anti- β-COP to the in vitro assay mix was found to block catalytic domain translocation from the lumen of endosomal vesicles in vitro. More recently, using a bioinformatic approach, Ratts et al. (2005) described the presence of a conserved motif, Tl

(TKIESLKEHG), in the transmembrane helix 1 of diphtheria toxin. Since the cytosolic expression of a peptide which carried the Tl motif in HuT102 cells was found to confer resistance to the toxin and knock down of peptide expression restored toxin sensitivity, it was apparent that either the Tl motif or transmembrane helix 1 of diphtheria toxin is likely to play an essential role in the catalytic domain entry process. In addition, pull down experiments using GST-DT 140-271 demonstrated that at least the β-COP subunit of the COPI complex specifically bound to at least a portion of the Tl motif, and that this association was also essential for catalytic domain entry process.

In the case of diphtheria toxin, the Tl motif includes two lysine residues which may play a role in COPI binding and catalytic domain entry; however, in anthrax lethal factor and anthrax edema factor the separation of their respective Tl- like motifs from the multiple upstream KXKXX COPI binding sequences raises addition questions as to the role that the Tl motif per se may play in the entry process. To address this question a site-directed mutational analysis of the multiple lysine residues in the N-terminal end of lethal factor was begun in order to determine the minimal sequence necessary for both COPI complex binding and delivery of

LFnDTA to the eukaryotic cell cytosol.

I have confirmed and extended these earlier observations and demonstrated that COPI binding to the transmembrane helix 1 is dependent upon the presence and spacing of at least three of the four lysine residues, two of which are positioned immediately upstream of the consensus Tl motif ( TKTKIESLKEHG). A site- directed mutational analysis of transmembrane helix 1 was first conducted in order to determine the minimal number of lysine residues that were necessary to facilitate catalytic domain delivery to the eukaryotic cell cytosol. Following site-directed mutagenesis and DNA sequence analysis to ensure the introduction of each mutation, individual mutant recombinant proteins were expressed, purified, and tested for cytotoxic activity by dose response analysis on HuT102 cells. With exception of a single mutation (K215A), all of the single K→ A mutant forms of DAI½<)IL-2 displayed only a modest 1 - 2-log reduction in their respective cytotoxic potency. In marked contrast, the introduction of double K→ A mutations {e.g., K213A, K215A or K215A, K217A) resulted in over a 5-log reduction in cytotoxic potency in their respective mutant fusion protein toxins. Taken together the results of this site- directed mutational analysis has led to the conclusion that a minimum of three lysine residues separated by three and four amino acids were required for full cytotoxic potency; whereas mutants that carried three lysine residues in transmembrane helix 1 that were separated by 1 and up to 6 amino acids retained cytotoxic activity; albeit with some having reduced levels of activity. In the first instance, the ε-amino groups of these lysine residues cluster on the same face of helical wheel projections; whereas, in the mutants with reduced activity the helical wheel projections of the ε-amino moieties show a greater distance along the vertical axis and distribution across the face (see Fig. 6). This observation suggests a degree of diversity of binding to COPI complex that is required for diphtheria toxin catalytic domain entry to the cytosol is somewhat greater than has been previously described for cargo and/or adaptor protein binding and trafficking (Eugster et al., 2004; Zerangue et al. 2001).

Hudson and Draper (1997) have previously demonstrated that at least two pairs of closely positioned amino moieties in neomycin were necessary to crosslink and induce precipitation of the COPI complex. In the present report, a family of synthetic peptides whose sequences were related to transmembrane helix 1 was used and it has been shown that the two KXKXX sequences carried by diphtheria toxin transmembrane helices 1 and 2 are also required to induce aggregation and precipitation of COPI complex in vitro. Since the addition of 1,3-cyclohexatiebis- methylamine (CBM) to the reaction mixture completely blocked COPI aggregation and precipitation in vitro, it is most likely that the interaction(s) between these toxin- related peptides and COPI complex is(are) mediated through free ε-amino moieties of the lysine residues within this region of the transmembrane domain. Indeed, a similar requirement was seen by Bethune et al. (2006) who defined the sequences from the cytoplasmic tail of the p23 adaptor protein that were required for COPI complex aggregation and precipitation. The COPI aggregation and precipitation in the presence of these synthetic peptides does not necessarily reflect the conditions necessary for catalytic domain entry. However, taken together these experiments suggest that the association between COPI and transmembrane helix 1 is likely to be mediated through the ε-amino moieties of lysine residues within this region of the transmembrane domain. In order to further examine the role of transmembrane helix 1 and/or the Tl motif in the catalytic domain entry process, it was reasoned that the functional ability to bind COPI complex per se rather than the native amino acid sequence of this portion of the toxin transmembrane domain was likely to be the critical factor for the translocation and delivery of the catalytic domain to the cytosol. To test this hypothesis, the transmembrane helix 1 was replaced with the 13 amino acid COPI binding sequence from the cytoplasmic tail region of the p23 adaptor protein. The resulting COPI binding domain swap mutant, DAB(212p23)₃₈9iL-2, was found to be as toxic as the wild type fusion protein toxin. It should be noted that the region of transmembrane helix 1 that was exchanged with p23 adaptor protein sequences contains all of the lysine residues that were shown to be essential for cytotoxic activity. The results presented here strongly suggest that at least one functional role of transmembrane helix 1 in diphtheria toxin is that of COPI complex binding, and that this protein-protein interaction is essential for the delivery of the catalytic domain to the eukaryotic cell cytosol.

While it has not been rigorously established which individual component(s) of the COPI complex mediates the interaction between the lysine-rich region of the diphtheria toxin transmembrane domain, the pull down of [³⁵S]-labeled γι-COP by GST-DT140-271 following in vitro transcription and translation suggests that this subunit along with β-COP (Ratts et al., 2005) is likely to participate in this interaction. The interaction(s) between transmembrane helix 1 of diphtheria toxin and COPI components appears to be unlike the interactions mediated by the canonical di- lysine signature KKXX with a-COP and β'-COP (Eugster et ah, 2004; Letourneur et ah, 1994). However, cargo and adaptor protein interactions with γ-COP have been reported to exhibit more diversity in their binding profile including KKXX and KXKXX motifs (Eugster et al., 2004; Zerangue et al. 2001), and results presented here suggest this diversity of binding may be extended to transmenbrane helices 1 and 2 of diphtheria toxin as well. Despite the relatively well characterized mechanism of COPI vesicle formation and function in retrograde traffic (for reviews see: Bethune et al, 2006; Nickel & Wieland, 2001), the role of COPI complex proteins in the trafficking of endosomal vesicles remains largely unknown.

The molecular process by which the catalytic domain from DAB₃₈9iL-2 is delivered to the eukaryotic cell cytosol appears to follow the following steps: (i) binding of the toxin to its respective cell surface receptor (Naglich et al, 1992), (ii) the internalization of the toxin: :receptor complex by receptor mediated endocytosis into an early endosomal compartment (Moya et al., 1985), (iii) upon acidification of the vesicle lumen by the (v)ATPase, the transmembrane domain spontaneously inserts into the endosomal vesicle membrane forming an 18 - 22A pore or channel (Donovan et al, 1981 ; Kagan et al., 1981 ). Once the a-carbon backbone of the toxin is "nicked" by the endoproteinase furin (Tsuneoka et al., 1993), the disulfide bond linked C- terminal of the catalytic domain and the N-terminal end of the transmembrane domain appear to be threaded in a fully denatured form into the pore formed by the membrane embedded transmembrane domain. As transmembrane helix 1 and the Tl motif emerge on the cytosolic surface of the vesicle membrane, COPI complex is likely to recognize these sequences as p23 adaptor and/or cargo-like cytoplasmic tail mimetics and bind to this portion of the transmembrane domain. However, unlike either the p23/p24 adaptor or cargo proteins which are anchored in the vesicle membrane, transmembrane helices 1-4 of the toxin with its disulfide bond linked catalytic domain appear to be un-tethered and readily "pulled" through the transmembrane vesicle pore formed by helices 5 - 9. Once the COPI complex facilitated delivery of the catalytic domain into the cytosol has occurred, it has been shown previously that thioredoxin reductase most likely reduces the disulfide bond between the N-terminal end of the transmembrane domain and the C-terminal end of the catalytic domain and at least Hsp90 appears to mediate refolding of the catalytic domain to a catalytically active ADP-ribosyltransferase (Ratts et al, 2003).

Experimental procedures

GST and GST-DT140-271 Pull-down experiments. GST and the fusion protein

GST-DT 140-271 expression and purification have been previously described by Ratts et al. (2003). Pull down experiments and HuT102 postnuclear supernatants were performed and prepared essentially as described by Tamayo et al. (2008).

Synthetic peptides. Peptides used in this study are listed in Table 3. All peptides were purchased from opella, Sudbury, MA and are amidated on their C-terminus.

The purity of all peptides as determined by high performance liquid chromatography was greater than 90%. Bovine liver COPI purification. COPI enriched fractions were prepared from bovine liver cytosol as described by Waters et al. (1991) with some modifications described by Tamayo et al. (2008). The 13S fraction containing intact COPI was further purified by DE52 (Whatman) column following manufacture's specifications in a Biologic LP (Bio-rad). Briefly, the DE52 cellulose was equilibrated with 25 mM Tris-HCl (pH 7.4)/ 100 mM KCl, 1 mM DTT, 10% Glycerol. The column was eluted with a step gradient of 150, 500, 750 and 1000 mM KCl in 25 mM Tris-HCl (pH 7.4)/ 10% Glycerol/ 1 mM DTT. The elution corresponding to 500 mM KCl containing intact COPI complex and associated material was dialyzed against 25 mM Tris.HCl (pH 7.4), 10% Glycerol and used as input material for the precipitation assays.

During all steps the different fractions were analyzed by immunoblot to confirm the presence of COPI components. Polyclonal antibodies to β-COP, γ-COP, ε-COP, ζ- COP were from Abeam and S.C. Biotechnologies.

In Vitro Synthesis of COPI subunits. Vector pCMV6-XL5 carrying the structural human gene encoding β' COP (NM_004766.1), γΙ-COP (NM 016128.3) and ε-COP (NM_007263.3) were purchased from OriGene Technologies (Rockville, MD). Full length COPI subunits were synthesized in vitro by using the TNT Quick Coupled Transcription/Translation System (Promega) in the presence of [³⁵S] -methionine following the manufacturer's instructions (2 μg of plasmid DNA/ 100 μΐ of reaction volume). After in vitro synthesis, the reaction mixture was diluted to 300 μΐ with binding buffer (50 mM Tris HC1, pH 7.4/150 mM NaCl/1 mM EDTA/1% Nonidet P- 40/ IX protease inhibitor cocktail (Roche)). Pull-down experiments with GST and GST-DT140-271 were performed as described above. Elutions were then analyzed by SDS/PAGE and autoradiographed according to standard methods.

COPI Precipitation assays. HuT102 cells postnuclear supernatant (25 μg of total protein) or partially purified COPI complex was incubated with increasing concentrations of CBM (Aldrich 180467) or the indicated concentrations of synthetic peptides in 40 μΐ of reaction buffer containing 25 mM Hepes KOH pH 7.4, 50 mM KCl, 25 mM Mg(OAc)₂ and IX Protease inhibitor cocktail (Roche) for two hours on ice. The mixture was centrifuged (13,000 x g for 25 min.) and the supernatant fluid and pellet fractions were probed for β-COP or yi-COP by immunoblot.

Bacterial strains, plasmids and fusion toxin products. The parental plasmid pET- JV127 (vanderSpek et al., 1993) encoding for the fusion toxin DAB₃₈₉IL-2 (AAA72359) was used for cloning and purification of the mutant toxins. The introduction of the alanine exchange mutations and the p23 adaptor COPI binding sequence swap was performed by site-directed mutagenesis between the Nsil and Rsill restriction sites (Table 4).

Table 4: Plasmids and IL-2 receptor-targeted fusion toxins used in this study.

Plasmid tox gene product amino acid sequence (212-225) pETJV127 DAB₃89lL-2 DKTKTKIESLKEHG pETCT20 DAB(K213A)₃₈₉IL-2 DATKTKIESLKEHG pETCT30 DAB(K215A)389IL-2 D TATKIESLKEHG pETCT40 DAB(K217A)3₈₉IL-2 DKTKTAIESLKEHG pETCT50 DAB(K222A)₃₈₉IL-2 DKTKTKIESLAEHG pETCT80 DAB(K213 A,K215 A)₃₈₉IL-2 DATATKIESLKEHG pETCT90 DAB(K215 A,K217A)₃₈₉lL-2 DKTATAIESLKEHG pETCT60 DAB(K213,K215,K217,K222- >A)₃₈₉IL-2 DATATAIESLAEHG pETCT70 DAB(212p23)₃₈₉IL-2 EIL KAKFFRREHG

Briefly, synthetic oligonucleotides containing the mutations were annealed and ligated into the previously digested parental vector. All mutations were confirmed by sequence analysis using the sequence primer 5' -GTCTCACTGAACCGTTGA-3'. All plasmids were propagated in chemically competent TOP 10 cells (Invitrogen) and protein induction was done in the HMS174 (DE3) (Novagen) strain as described by vanderSpek et al. (1993). The synthetic oligonucleotides sequences used in this study are available upon request.

Purification of toxin mutant proteins. The induction of expression, inclusion body preparation and protein purification was performed using a protocol previously reported by vanderSpek et al. (1993). Proteins were analyzed by SDS-PAGE electrophoresis, 7% Native gel electrophoresis and immunoblot (Mo Anti DTA, Abeam). Protein concentrations were determined by the modified Bradford reagent (Pierce). The percent full length toxin in each preparation was determined by calculating the area under the curve corresponding to toxin of silver stained 7% SDS- PAGE using Scion Image for Windows software.

Cytotoxicity Assay. Cytotoxicity assays were performed as described by vanderSpek et al. (1994). Figures were created in GraphPad Prism version 5.01 for Windows, GraphPad Software, San Diego California USA. References

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All publications and patents cited in this specification, including U.S. Provisional Appliation Serial No. 61/327,024, are hereby incorporated by reference herein as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

What is claimed is:

Claims

1. A conjugate compound of the invention having the general formula:

X-Y-Z (Formula I), wherein:

X is selected from a cytotoxic agent, a therapeutic agent, and a diagnostic agent and comprises at least one thiol-containing group capable of forming a disulfide bond with Y;

Z is a polypeptide targeting moiety that is bound to Y at its carboxy-terminal end.

2. The compound of claim 1, wherein said X is selected from a siRNA, dsRNA, an RNAi molecule, a protein nucleic acid (PNA) molecule, and a

polypeptide.

3. The compound of claim 2, wherein said polypeptide is a transcription factor or growth factor.

4. The compound of any one of claims 1 to 3, wherein said lysine-rich domain comprises one or more lysine rich motifs having a dibasic signature selected from KKXX and KXKXX, or aromatic amino acid sequences selected from FFXXBB(X)„,.

5. The compound of any one of claims 1 to 4, wherein Y comprises an amino acid sequence having at least 80% sequence identity to a contiguous amino acid sequence corresponding to at least amino acids 201 to 235 of diphtheria toxin.

6. The compound of claim 5, wherein Y comprises an amino acid sequence having at least 80% sequence identity to a contiguous amino acid sequence corresponding to amino acids 195 to 389 of diphtheria toxin.

7. The compound of claim 6, wherein Y comprises amino acids 195 to 389 of diphtheria toxin.

8. The compound of any one of claims 1 to 7, wherein said thiol-containing group of X is a cysteine residue that forms a disulfide bond with a cysteine residue of Y.

9. The compound of any one of claims 1 to 8, wherein X is a nucleic acid molecule and said thiol-containing group is located at the 3' or 5' end of X, or wherein X is a polypeptide and said thiol-containing group is located at the amino- or carboxy- terminal end of X.

10. The compound of any one of claims 1 to 9, wherein Z is selected the group consisting of insulin, insulin-like growth factor receptor 1 (IGF1R), IGF2R, insulin-like growth factor (IGF; e.g., IGF 1 or 2), mesenchymal epithelial transition factor receptor (c-met; also known as hepatocyte growth factor receptor (HGFR)), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), heregulin, fibroblast growth factor receptor (FGFR), platelet- derived growth factor receptor (PDGFR), platelet-derived growth factor (PDGF), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor (VEGF), tumor necrosis factor receptor (TNFR), tumor necrosis factor alpha (TNF-a), TNF-β, folate receptor (FOLR), folate, transferring, transferrin receptor (TfR), mesothelin, Fc receptor, c-kit receptor, c-kit, an integrin (e.g., an a4 integrin or a β-l integrin), P-sclcctin, sphingosine-1 -phosphate receptor- 1 (SI PR), hyaluronate receptor, leukocyte function antigen-1 (LFA-1), CD4, CD11, CD18, CD20, CD25, CD27, CD52, CD70, CD80, CD85, CD95 (Fas receptor), CD 106 (vascular cell adhesion molecule 1 (VCAMl), CD166 (activated leukocyte cell adhesion molecule (ALCAM)), CD178 (Fas ligand), CD253 (TNF-related apoptosis-inducing ligand (TRAIL)), ICOS ligand, CCR2, CXCR3, CCR5, CXCL12 (stromal cell-derived factor 1 (SDF-1)), interleukin 1 (IL-1), IL-lra, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, CTLA-4, MART-1, gplOO, MAGE-1, ephrin (Eph) receptor, mucosal addressin cell adhesion molecule 1 (MAdCAM-1), carcinoembryonic antigen (CEA), Lewis^Y, MUC-1, epithelial cell adhesion molecule (EpCAM), cancer antigen 125 (CA125), prostate specific membrane antigen (PSMA), TAG-72 antigen, erythroblastic leukemia viral oncogene homolog (ErbB) receptor, and fragments thereof.

11. The compound of any one of claims 1 to 10, wherein Z is an antibody.

12. A method of treating disease in a mammal in need thereof by administering the conjugate compound of any one of claims 1 to 11 to said mammal, wherein said conjugate compound comprises a cytotoxic or therapeutic agent as X.

13. A method of diagnosing disease in a mammal by administering the conjugate compound of any one of claims 1 to 11 to said mammal, wherein said conjugate compound comprises a detectable label as X.

14. The method of claim 12 or 13, wherein said mammal is a human.